Cancer:
Breast, kidney, prostate, renal., liver, endometrial., ovarian

Action: Anti-angiogenesis, cell-cycle arrest, cancer stem cells, VEGF, radiotherapy, sex hormone-binding globulin (SHBG), insulin-like growth factor-1 (IGF-1)

Genistein is a natural isoflavone phytoestrogen present in a number of plants, including soy, fava, and kudzu (Glycine max [(L.) Merr.], Vicia faba (L.), Pueraria lobata [(Willd.) Ohwi]).

Phytoestrogens

Phytoestrogens have been investigated at the epidemiological., clinical and molecular levels to determine their potential health benefits. The two major groups of phytoestrogens, isoflavones and lignans, are abundant in soy products and flax respectively, but are also present in a variety of other foods. It is thought that these estrogen-like compounds may protect against chronic diseases, such as hormone-dependent cancers, cardiovascular disease and osteoporosis (Stark & Madar, 2002).

S-Equol Production and Isoflavone Metabolism

S-Equol and Breast Cancer

Differences in ability to metabolize daidzein to equol might help explain inconsistent findings about isoflavones and breast cancer. Tseng et al. (2013) examined equol-producing status in relation to breast density, a marker of breast cancer risk, and evaluated whether an association of isoflavone intake with breast density differs by equol-producing status in a sample of Chinese immigrant women. In their sample, 30% were classified as equol producers. In adjusted linear regression models, equol producers had significantly lower mean dense tissue area (32.8 vs. 37.7 cm(2), P = 0.03) and lower mean percent breast density (32% vs. 35%, P = 0.03) than nonproducers. Significant inverse associations of isoflavone intake with dense area and percent density were apparent, but only in equol producers (interaction P = 0.05 for both).

Although these findings warrant confirmation in a larger sample, they offer a possible explanation for the inconsistent findings about soy intake and breast density and possibly breast cancer risk as well. The findings further suggest the importance of identifying factors that influence equol-producing status and exploring appropriate targeting of interventions.

S-Equol and Dietary Factors

S-(-)equol, an intestinally derived metabolite of the soy isoflavone daidzein, is proposed to enhance the efficacy of soy diets. Setchell et al. (2013) performed a comprehensive dietary analysis of 143 macro- and micro-nutrients in 159 healthy adults to determine whether the intake of specific nutrients favors equol production. Three-day diet records were collected and analyzed using Nutrition Data System for Research software and S-(-)equol was measured in urine by mass spectrometry.

Equol producers accounted for 29.6% of participants. No significant differences were observed for total protein, carbohydrate, fat, saturated fat, or fiber intakes between equol producers and nonproducers. However, principal component analysis revealed differences in several nutrients, including higher intakes of polyunsaturated fatty acids (P = 0.039), maltose (P = 0.02), and vitamins A (P = 0.01) and E (P = 0.035) and a lower intake of total cholesterol (P = 0.010) in equol producers.

Subtle differences in some nutrients may influence the ability to produce equol.

S-Equol and Dietary Factors; Fats

The soy isoflavones, daidzein and genistein, and the lignans, matairesinol and secoisolariciresinol, are phytoestrogens metabolized extensively by the intestinal microflora. Considerable important evidence is already available that shows extensive interindividual variation in isoflavone metabolism. There was a 16-fold variation in total isoflavonoid excretion in urine after the high-isoflavone treatment period. The variation in urinary equol excretion was greatest (664-fold), and subjects fell into two groups: poor equol excretors and good equol excretors (36%). A significant negative correlation was found between the proportion of energy from fat in the habitual diet and urinary equol excretion (r = -0.55; p = 0.012). Good equol excretors consumed less fat as percentage of energy than poor excretors (26 +/- 2.3% compared with 35 +/- 1.6%, p < 0.01) and more carbohydrate as percentage of energy than poor excretors (55 +/- 2.9% compared with 47 +/- 1.7%, p < 0.05).

It is suggested that the dietary fat intake decreases the capacity of gut microbial flora to synthesize equol (Rowland et al., 2000).

Isoflavones and Fermented Soy Foods

Serum concentrations of total isoflavones after 1–4 hours were significantly higher in the aglycone-rich fermented soybeans (Fsoy) group than in the glucoside-rich non-fermented soybeans (Soy) group. The Fsoy group showed significantly higher maximum concentration (Cmax: 2.79 ± 0.13 vs 1.74 ± 0.13 µmol L(-1) ) and area under the curve (AUC(0-24 h) : 23.78 ± 2.41 vs 19.95 ± 2.03 µmol day L(-1) ) and lower maximum concentration time (Tmax: 1.00 ± 0.00 vs 5.00 ± 0.67 h) compared with the Soy group. The cumulative urinary excretion of total isoflavones after 2 hours was significantly higher in the Fsoy group than in the Soy group. Individual isoflavones (daidzein, genistein and glycitein) showed similar trends to total isoflavones. Equol (a metabolite from daidzein) did not differ between the two groups.

The results of this study demonstrated that the isoflavones of aglycone-rich Fsoy were absorbed faster and in greater amounts than those of glucoside-rich Soy in postmenopausal Japanese women (Okabe et al., 2011).

Phytoestrogens and Breast Cancer; ER+/ER-, ER α /ER β

Dietary-derived Anti-angiogenic Compounds

Consumption of a plant-based diet can prevent the development and progression of chronic diseases that are associated with extensive neovascularization; however, little is known about the mechanisms. To determine whether prevention might be associated with dietary-derived angiogenesis inhibitors, the urine of healthy human subjects consuming a plant-based diet was fractionated and the fractions examined for their ability to inhibit the proliferation of vascular endothelial cells.

The isoflavonoid genistein was the most potent, and inhibited endothelial cell proliferation and in vitro angiogenesis at concentrations giving half-maximal inhibition of 5 and 150 microM, respectively. Genistein concentrations in urine of subjects consuming a plant-based diet are in the micromolar range, while those of subjects consuming a traditional Western diet are lower by a factor of > 30. The high excretion of genistein in urine of vegetarians and in addition to these results suggest that genistein may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors, by inhibiting neovascularization.

Thus, genistein may represent a member of a new class of dietary-derived anti-angiogenic compounds (Fotsis et al., 1993).

ERβ as a Down-regulator of ER+ Breast Cancer

The estrogen receptor (ER) isoform known as ERβ has become the focus of intense investigation as a potential drug target. The existence of clear-cut differences in ERβ and ERα expression suggests that tissues could be differentially targeted with ligands selective for either isoform (Couse et al., 1997; Enmark et al., 1997). In particular, the fact that ER β is widely expressed but not the primary estrogen receptor in, for example, the uterus (where estrogenic effects are mediated via ERα) (Harris, Katzenellenbogen, & Katzenellenbogen, 2002) opens up the possibility of targeting other tissues while avoiding certain classical estrogenic effects.

A major advance toward understanding how some phytoestrogens achieve modest ERβ selectivity was the X-ray structure determination of the ERβ ligand binding domain (LBD) complexed with genistein (GEN) (Pike et al., 1999), a 40-fold ERβ-selective ligand (Harris et al., 2002). This study clearly showed that there are only two residue substitutions in close proximity to GEN: ERα Leu384 is replaced by ER β Met336, and ERα Met421 is replaced by ER β Ile373.

ERbeta works as counter partner of ERalpha through inhibition of the transactivating function of ERalpha by heterodimerization, distinct regulation on several specific promoters by ERalpha or ERbeta, and ERbeta-specific regulated genes which are probably related to its anti-proliferative properties. Epidemiological studies of hormone replacement therapy and isoflavone (genistein) consumption indicate the possible contribution of ERbeta-specific signaling in breast cancer prevention. A selective estrogen receptor modulator, which works as an antagonist of ERalpha and an agonist of ERbeta, may be a promising chemo-preventive treatment (Saji, Hirose, & Toi, 2005).

Genistein and Apoptosis

The association between consumption of genistein containing soybean products and lower risk of breast cancer suggests a cancer chemo-preventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF-7 and MDA-MB-231 human breast cancer cells that express the wild-type and mutant p53 gene, respectively.

After 6 days of incubation with 50 microM genistein, MCF-7, but not MDA-MB-231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly-(ADP-ribose)-polymerase and significant DNA fragmentation were also detected in MCF-7 cells.

In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF-7, but not MDA-MB-231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was also studied. Levels of p21 increased in both of the genistein-treated cell lines, suggesting that p21 gene expression was activated but in a p53-independent manner; whereas no significant changes in levels of the pro-apoptotic protein, Bax, were found. In MCF-7 cells, levels of the anti-apoptotic protein, Bcl-2, decreased slightly at 18–24 hours but then increased considerably after 48 hours. Hence, the Bax:Bcl-2 ratio initially increased but later decreased.

Data suggests that at the concentration tested, MCF-7 cells, in contrast to MDA-MB-231 cells, were sensitive to the induction of apoptosis by genistein. However, the roles of Bax and Bcl-2 are unclear (Xu & Loo, 2001).

Genistein Derivatives and Breast Cancer Inhibition

Genistein binds to estrogen receptors and stimulates growth at concentrations that would be achieved by a high soy diet, but inhibits growth at high experimental concentrations.

The estrogen receptor (ER) is a major target for the treatment of breast cancer cells. Genistein, a soy isoflavone, possesses a structure similar to estrogen and can both mimic and antagonize estrogen effects although at high concentrations it inhibits breast cancer cell proliferation. Hence, to enhance the anti-cancer activity of Genistein at lower concentrations, seven structurally modified derivatives of Genistein based on the structural requirements for an optimal anti-cancer effect were synthesised. Among those seven, three derivatives showed high anti-proliferative activity with IC(50) levels in the range of 1-2.5 µM, i.e., at much lower concentrations range than Genistein itself, in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) studied. In our analysis, we noticed that at IC(50) concentrations, the MA-6, MA-8 and MA-19 Genistein derivatives induced apoptosis, inhibited ER-α messenger RNA expression and increased the ratio of ER-β to ER-α levels in a manner comparable to that of the parent compound Genistein.

Of note, these three modified Genistein derivatives exerted their effects at concentrations 10–15 times lower than the parent compound, decreasing the likelihood of significant ER- α pathway activation, which has been a concern for Genistein. Hence these compounds might play a useful role in breast cancer chemoprevention (Marik et al., 2011).

Genistein and ER α

To determine the effects of low-dose, long-term genistein exposure MCF-7 breast cancer cells were cultured in 10nM genistein for 10-12 weeks and investigated whether or not this long-term genistein treatment (LTGT) altered the expression of estrogen receptor alpha (ERalpha) and the activity of the PI3-K/Akt signaling pathway. This is known to be pivotal in the signaling of mitogens such as oestradiol (E(2)), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). LTGT significantly reduced the growth promoting effects of E(2) and increased the dose-dependent growth-inhibitory effect of the PI3-K inhibitor, LY 294002, compared to untreated control MCF-7 cells.

This was associated with a significant decreased protein expression of total Akt and phosphorylated Akt but not ERalpha. Rapamycin, an inhibitor of one of the downstream targets of Akt, mammalian target of rapamycin (mTOR), also dose-dependently inhibited growth but the response to this drug was similar in LTGT and control MCF-7 cells. The protein expression of liver receptor homologue-1 (LRH1), an orphan nuclear receptor implicated in tumorigenesis was not affected by LTGT.

These results show that LTGT results in a down-regulation of the PI3-K/Akt signaling pathway and may be a mechanism through which genistein could offer protection against breast cancer (Anastasius et al., 2009).

Genistein and ER+/ER-

Genistein was found to cause a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 M genistein showed a dose-dependent accumulation in the G2M phase of the cell-cycle. At the highest tested concentration, there was a 7-fold increase in the percentage of cells in G2M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate 4-fold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G2M arrest was coupled with a parallel depletion of the G0/G1 phase.

To understand the mechanism of action underlying the block in G2M induced by genistein, Cappelletti et al. (2000) investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G2M transition. As expected, p34cdc-2 expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With the exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34cdc-2 catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a 4-fold decrease in kinase activity evaluated using the histone H1 as substrate.

Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 M genistein, cyclin B1 (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells.

We therefore conclude that genistein causes a G2M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34cdc-2/cyclin B1 complex only in all of the studied cell lines.

Genistein and ER+/ER-; MDR

Genistein is a potent inhibitor of the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive) (IC50 values from 6.5 to 12.0 µg/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by over expression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells (Peterson et al., 1991).

Studies have shown that genistein exerts multiple suppressive effects on both estrogen receptor positive (ER+) as well as estrogen receptor negative (ER-) human breast carcinoma lines suggesting that the mechanisms of these effects may be independent of ER pathways.

In the present study however Shao et al. (2000) provide evidence that in the ER+ MCF-7, T47D and 549 lines but not in the ER-MDA-MB-231 and MDA-MB-468 lines both presumed 'ER-dependent' and 'ER-independent' actions of genistein are mediated through ER pathways. Genistein's anti-proliferative effects are estrogen dependent in these ER+ lines, being more pronounced in estrogen-containing media and in the presence of exogenous 17-beta estradiol. Genistein also inhibits the expression of ER-downstream genes including pS2 and TGF-beta in these ER+ lines and this inhibition is also dependent on the presence of estrogen. Genistein inhibits estrogen-induced protein tyrosine kinase (PTK) activity. Genistein is only a weak transcriptional activator and actually decreases ERE-CAT levels induced by 17-beta estradiol in the ER+ lines.

Genistein also decreases steady state ER mRNA only in the presence of estrogen in the ER+ lines thereby manifesting another suppression of and through the ER pathway. Their observations resurrect the hypothesis that genistein functions as a 'good estrogen' in ER+ breast carcinomas. Since chemo-preventive effects of genistein would be targeted to normal ER-positive ductal-lobular cells of the breast, this 'good estrogen' action of genistein is most relevant to our understanding of chemoprevention.

Genistein and Concentration

The anti-proliferative activity of the isoflavones daidzein and genistein were investigated in three breast cancer cell lines with different patterns of estrogen receptor (ER) and c erbB 2 protein expression (ERα positive MCF 7 cells, c erbB 2 positive SK BR 3 cells and ERα/c erbB 2 positive ZR 75 1). After treatment at various concentrations (1 200 µM for 72 hours), the effect of daidzein and genistein on the proliferation of different cell types varied; these effects were found to be associated with ERα and c erbB 2 expression. Daidzein and genistein exhibited biphasic effects (stimulatory or inhibitory) on proliferation and ERα expression in MCF 7 cells. Although 1 µM daidzein significantly stimulated cell growth, ERα expression was unaffected. However, genistein showed marked increases in proliferation and ERα expression after exposure to <10 µM genistein.

Notably, the inhibition of cell proliferation by 200 µM genistein was greater compared to that by daidzein at the same concentration. Daidzein and genistein significantly inhibited proliferation of SK BR 3 and ZR 75 1 cells in a dose-dependent manner. In addition, ERα and c erbB 2 expression was reduced by daidzein and genistein in both SK BR 3 and ZR 75 1 cells in a dose-dependent manner. However, the effect of genistein was greater compared to that of daidzein.

In conclusion, the isoflavones daidzein and genistein showed anti breast cancer activity, which was associated with expression of the ERα and c erbB 2 receptors (Choi et al., 2013).

ER- α / ER β Receptors

Isoflavones are phytoestrogens that have been linked to both beneficial as well as adverse effects in relation to cell proliferation and cancer risks. The mechanisms that could be involved in this dualistic mode of action were investigated. One mechanism relates to the different ultimate cellular effects of activation of estrogen receptor (ER) α, promoting cell proliferation, and of ERβ, promoting apoptosis, with the major soy isoflavones genistein and daidzein activating especially ERβ.

A second mode of action includes the role of epigenetics, including effects of isoflavones on DNA methylation, histone modification and miRNA expression patterns. The overview presented reveals that we are only at the start of unraveling the complex underlying mode of action for effects of isoflavones, both beneficial or adverse, on cell proliferation and cancer risks. It is evident that whatever model system will be applied, its relevance to human tissues with respect to ERα and ERβ levels, co-repressor and co-activator characteristics as well as its relevance to human exposure regimens, needs to be considered and defined (Rietjens et al., 2013).

Genistein and ER+/ER-, ER- α / ER β Receptors

A novel mechanism of adipokine, adiponectin (APN) -mediated signaling that influences mammary epithelial cell proliferation, differentiation, and apoptosis to modify breast cancer risk has been identified. It was demonstrated that early dietary exposure to soy protein isolate induced mammary tissue APN production without corresponding effects on systemic APN levels. In estrogen receptor (ER)-negative MCF-10A cells, recombinant APN promoted lobuloalveolar differentiation by inhibiting oncogenic signal transducer and activator of transcription 3 activity.

In ER-positive HC11 cells, recombinant APN increased ERβ expression, inhibited cell proliferation, and induced apoptosis. Using the estrogen-responsive 4X-estrogen response element promoter-reporter construct to assess ER transactivation and small interfering RNA targeting of ERα and ERβ, Rahal et al. (2011) show that APN synergized with the soy phytoestrogen genistein to promote ERβ signaling in the presence of estrogen (17β-estradiol) and ERβ-specific agonist 2,3-bis(4-hydroxyphenyl)-propionitrile and to oppose ERα signaling in the presence of the ERα-specific agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol.

The enhancement of ERβ signaling with APN + genistein co-treatments was associated with induction of apoptosis, increased expression of pro-apoptotic/prodifferentiation genes (Bad, p53, and Pten), and decreased anti-apoptotic (Bcl2 and survivin) transcript levels. These results suggest that mammary-derived APN can influence adjacent epithelial function by ER-dependent and ER-independent mechanisms that are consistent with reduction of breast cancer risk and suggest local APN induction by dietary factors as a targeted approach for promotion of breast health.

Genistein and Non-breast Cancer

Genistein Concentrations; Endometrial Cancer

The influence of two phytoestrogens (Genistein and Daidzein) on estrogen-related receptor-α in endometrial cancer cell line Ishikawa was investigated on the proliferation of the cells in this cell line. Ishikawa cells were incubated with different concentrations of Genistein and Daidzein (40, 20, 10, 5 µmol/L) for 24 hours or 48 hours, followed by Real-Time PCR for analyzing the expression of ERR-α mRNA in the cell line. MTT assay was then performed to evaluate the proliferation of Ishikawa cells.

The expression level of ERR-α mRNA in Ishikawa cells was higher than that of the control group after being dealt for 24 hours or 48 hours with Genistein, and the concentration 20 µmol/L was most effective. Nevertheless, this up-regulation was blocked when the cells were treated with 40 µmol/L Genistein. Lower concentration (5, 10 µmol/L) Genistein had depressant effect on proliferation of the cells, while higher concentrations (20, 40 µmol/L) had stimulant effect. After being treated with different concentrations of Daidzein, the expression of ERR- α mRNA in all experimental groups was significantly higher than that in the control group. In the 24 hour group, the concentration 40 µmol/L had most obvious effect; but in the 48 hour group, the concentration 20 µmol/L had most obvious effect, and this up-regulation was blocked when the concentration was elevated to 40 µmol/L.

Noticeably, all concentrations of Daidzein had depressant effect on the proliferation of Ishikawa cells in both 24 hour and 48 hour groups. In the 24 hour group, lower concentrations were more effective, but in the 48 hour group, concentration showed no significant effect. In lower concentrations, both Genistein and Daidzein have up-regulation effect on the expression of ERR-α, and block the proliferation of Ishikawa cells; but in higher concentrations, the up-regulation effect on ERR-α mRNA expression by these two phytoestrogens is not obvious. Genistein stimulates the proliferation of lshikawa cells in higher concentrations, while Daidzein suppresses the proliferation, especially in lower concentrations (Xin et al., 2009).

Genistein and VEGF; Ovarian Cancer

Genistein represses NF-kappaB (NF-κB), a pro-inflammatory transcription factor, and inhibits pro-inflammatory cytokines such as TNF-α and IL-6 in epithelial ovarian cancer. Additionally, it has been shown to stabilize p53 protein, sensitize TRAIL (TNF receptor apoptosis-inducing ligand) induce apoptosis, and prevent or delay chemotherapy-resistance. Recent studies further indicate that genistein potently inhibits VEGF production and suppresses ovarian cancer cell metastasis in vitro.

Based on widely published in vitro and mouse-model data, some anti-inflammatory phytochemicals appear to exhibit activity in modulating the tumor microenvironment. Specifically, apiegenin, baicalein, curcumin, EGCG, genistein, luteolin, oridonin, quercetin, and wogonin repress NF-kappaB (NF-κB, a pro-inflammatory transcription factor) and inhibit pro-inflammatory cytokines such as TNF-α and IL-6. Recent studies further indicate that apigenin, genistein, kaempferol, luteolin, and quercetin potently inhibit VEGF production and suppress ovarian cancer cell metastasis in vitro. Lastly, oridonin and wogonin were suggested to suppress ovarian CSCs as is reflected by down-regulation of the surface marker EpCAM (Chen, Michael, & Butler-Manuel, 2012).

Renal Cell Carcinoma, Prostate Cancer; Radiotherapy

The KCI-18 RCC cell line was generated from a patient with papillary renal cell carcinoma. Tumor cells metastasize from the primary renal tumor to the lungs, liver and mesentery mimicking the progression of RCC in humans. Treatment of established kidney tumors with genistein demonstrated a tendency to stimulate the growth of the primary kidney tumor and increase the incidence of metastasis to the mesentery lining the bowel. In contrast, when given in conjunction with kidney tumor irradiation, genistein significantly inhibited the growth and progression of established kidney tumors. These findings confirm the potentiation of radiotherapy by genistein in the orthotopic RCC model as previously shown in orthotopic models of prostate cancer. These studies in both RCC and prostate tumor models demonstrate that the combination of genistein with primary tumor irradiation is a more effective and safer therapeutic approach as the tumor growth and progression are inhibited both in the primary and metastatic sites (Gilda et al., 2007).

Cell-cycle Arrest

Genistein treatment increased Wee1 levels and decreased phospho-Wee1 (Ser 642). Moreover, genistein substantially decreased the Ser473 and Thr308 phosphorylation of Akt and up-regulated PTEN expression. Down-regulation of PTEN by siRNA in genistein-treated cells increased phospho-Wee1 (Ser642), whereas it decreased phospho-Cdc2 (Tyr15), resulting in decreased G2/M cell-cycle-arrest. Therefore, induction of G2/M cell-cycle arrest by genistein involved up-regulation of PTEN (Liu et al., 2013).

Cancer Stem Cells (CSCs)

Cancer stem cells (CSCs) are cells that exist within a tumor with a capacity for self-renewal and an ability to differentiate, giving rise to heterogeneous populations of cancer cells. These cells are increasingly being implicated in resistance to conventional therapeutics and have also been implicated in tumor recurrence. Several cellular signaling pathways including Notch, Wnt, phosphoinositide-3-kinase-Akt-mammalian target of rapamycin pathways, and known markers such as CD44, CD133, CD166, ALDH, etc. have been associated with CSCs.

Here, we have reviewed our current understanding of self-renewal pathways and factors that help in the survival of CSCs with special emphasis on those that have been documented to be modulated by well characterized natural agents such as curcumin, sulforaphane, resveratrol, genistein, and epigallocatechin gallate (Dandawate et al., 2013).

Genistein and Sex Hormone-binding Globulin (SHBG)

Studies have indicated a correlation between a high level of urinary lignans and isoflavonoid phytoestrogens, particularly genistein, and a low incidence of hormone-dependent cancers, such as breast and prostate cancer. Previously it has been observed that a vegetarian diet is associated with high plasma levels of sex hormone-binding globulin (SHBG), reducing clearance of sex hormones and probably risk of breast and prostate cancer. In the present study we investigated the in vitro effect of genistein on the production of SHBG by human hepatocarcinoma (Hep-G2) cells in culture and its effect on cell proliferation.

It has additionally been found that genistein not only significantly increases the SHBG production by Hep-G2 cells, but also suppresses the proliferation of those cancer cells already at a stage when SHBG production continues to be high. It is hence concluded that, in addition to the lignan enterolactone, the most abundant urinary isoflavonoid genistein stimulates SHBG production and inhibits Hep-G2 cancer cell proliferation (Mousavi et al., 1993).

Insulin-like Growth Factor-1 (IGF-1); Prostate Cancer

Elevated levels of insulin-like growth factor-1 (IGF-1) are associated with an increased risk of several different cancers, including prostate cancer. Inhibition of IGF-1 and the downstream signaling pathways mediated by the activation of the IGF-1 receptor (IGF-1R) may be involved in inhibiting prostate carcinogenesis. Genistein treatment caused a significant inhibition of IGF-1-stimulated cell growth. Flow cytometry analysis revealed that genistein significantly decreased the number of IGF-1-stimulated cells in the G0/G1 phase of the cell-cycle. In IGF-1-treated cells, genistein effectively inhibited the phosphorylation of IGF-1R and the phosphorylation of its downstream targets, such as Src, Akt, and glycogen synthase kinase-3β (GSk-3β). IGF-1 treatment decreased the levels of E-cadherin but increased the levels of β-catenin and cyclin D1.

However, genistein treatment greatly attenuated IGF-1-induced β-catenin signaling that correlated with increasing the levels of E-cadherin and decreasing cyclin D1 levels in PC-3 cells. In addition, genistein inhibited T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity. These results showed that genistein effectively inhibited cell growth in IGF-1-stimulated PC-3 cells, possibly by inhibiting downstream of IGF-1R activation (Lee et al., 2012).

Sex Hormone-binding Globulin (SHBG); Hepatoma

Sex hormone-binding globulin (SHBG) is the main transport binding protein for sex steroid hormones in plasma and regulates their accessibility to target cells. Plasma SHBG is secreted by the liver under the control of hormones and nutritional factors. In the human hepatoma cell line (HepG2), thyroid and estrogenic hormones, and a variety of drugs including the anti-estrogen tamoxifen, the phytoestrogen, genistein and mitotane (Op'DDD) increase SHBG production and SHBG gene promoter activity. In contrast, monosaccharides (glucose or fructose) effectively decrease SHBG expression by inducing lipogenesis, which reduces hepatic HNF-4alpha levels, a transcription factor that plays a critical role in controlling the SHBG promoter. Interestingly, diminishing hepatic lipogenesis and free fatty acid liver biosynthesis also appear to be associated with the positive effects of thyroid hormones and PPARgamma antagonists on SHBG expression.

This mechanism provides a biological explanation for why SHBG is a sensitive biomarker of insulin resistance and the metabolic syndrome, and why low plasma SHBG levels are a risk factor for developing hyperglycemia and type 2 diabetes, especially in women (Pugeat et al., 2009).

Cancer: Pancreatic

Pancreatic cancer remains the fourth most common cause of cancer related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of pancreatic cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on pancreatic cancer cells has not been fully elucidated.

Wang et al., (2006) have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway. Moreover, Notch signaling is known to play a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of pancreatic cancer. Hence, in our study, they investigated whether there is any cross talk between Notch and NF-kappaB during genistein-induced apoptosis in BxPC-3 pancreatic cancer cells. They found that genistein inhibits cell growth and induces apoptotic processes in BxPC-3 pancreatic cancer cells.

This was partly due to inhibition of Notch-1 activity. BxPC-3 cells transfected with Notch-1 cDNA showed induction of NF-kappaB activity, and this was inhibited by genistein treatment. From these results, we conclude that the inhibition of Notch-1 and NF-kappaB activity and their cross talk provides a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in pancreatic cancer cells.

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