Molecular Effects of the Isoflavonoid Genistein in Prostate Cancer

Translational Medicine

Molecular Effects of the Isoflavonoid Genistein in Prostate Cancer Jasmin Bektic Roman Guggenberger Iris E. Eder Alexandre E. Pelzer Andreas P. Berger Georg Bartsch Helmut Klocker

Abstract Differences in diet have been proposed to be at least partially responsible for the low rate of prostate cancer in Asian populations compared with men in Western countries. One of the compounds that occurs in a greater quantity in the Eastern diet is genistein, an isoflavonoid found in high concentrations in serum after ingestion of soy-rich foods. Extensive molecular studies have been performed to determine its potential health benefits. The mechanism of action of genistein is complex and includes several cellular pathways. In addition to its estrogenic and/or antiestrogenic activities, genistein has been reported to inhibit steroidogenesis and block several protein tyrosine kinases, including epidermal growth factor receptor and src tyrosine kinases. Moreover, it arrests the cell cycle, induces apoptosis, and has antiangiogenic and antimetastatic properties and antioxidant activity. Herein, we review the current literature on the molecular mechanisms of genistein in relation to its effects on prostate cancer cells.

Department of Urology, Innsbruck Medical University, Austria

Introduction Phytoestrogens are plant-derived nonsteroidal polyphenolic compounds that structurally or functionally mimic mammalian estrogens and show potential benefits for human health. The estrogenic properties of certain plants have been recognized for > 50 years. In the mid-1940s, an infertility syndrome in sheep had been attributed to the ingestion of clover containing high levels of the isoflavones formononetin and biochanin A.1 More recently, an increasing number of epidemiologic and experimental studies have suggested that the consumption of phytoestrogen-rich foods may have protective effects on estogen-related conditions, such as menopausal symptoms,2 osteoporosis,3 and cardiovascular diseases,4 and on hormone-related diseases such as prostate5 and breast cancer.6 Many women turn to phytoestrogens as an alternative to hormone replacement therapy because of the undesirable side effects of hormonal therapy such as increased risk of breast and endometrial cancer and irregular bleeding.7 However, concerns have been raised about the potential dangers of consuming high levels of these compounds.8 In principle, phytoestrogens do not act differently from synthetic compounds and thus may bear the same risk of side effects. Consequently, phytoestrogens are currently under active investigation for their roles in human health.9

Clinical Prostate Cancer, Vol. 4, No. 2, 124-129, 2005 Key words: Apoptosis, Cell cycle, Phytoestrogen, Steroid hormones Submitted: Dec 14, 2004; Revised: Apr 14, 2004; Accepted: May 12, 2005 Address for correspondence: Helmut Klocker, PhD Department of Urology Innsbruck Medical University Anichstr 35 6020 Innsbruck, Austria Fax: 43-512-504-6724818 e-mail: [email protected]

Uptake and Metabolism of Genistein

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Isoflavones are the most studied group of phytoestrogens and are found almost exclusively in the family of Leguminosae. Soybeans are a rich source of isoflavones and contain approximately 2 grams of isoflavones per kilogram fresh weight.10 A large numer of isoflavones have been identified from plants. Their principal representatives are daidzein and genistein. They occur in plants as the inactive glycosides daidzin and genistin and their 4'-methyl ether derivates, formononetin and biochanin A. The β-glycosides are heat-sta-

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ble and survive cooking.11 In the digestive tract, these precursors can be metabolized by the enzymes of the normal microflora, and their corresponding aglycones, genistein and daidzein, are released.12,13 The gastrointestinal microflora can further metabolize daidzein to the potent phytoestrogen equol, but this biotransformation has a high interindividual variability.14 Another metabolite produced from daidzein is O-desmethylangolensin. Metabolism of genistein by the microflora yields the end products 2-(4-hydroxyphenyl) propanoic acid and 1,3,5-trihydroxybenzene15,16 (Figure 1). A recent study showed that isoflavone glycosides were not absorbed intact across the enterocyte of healthy adults, and thus hydrolysis of the sugar residue was required for the absorption of isoflavones.16 Because of high soy intake, Asian subjects have 5-100 times higher serum levels of isoflavonoids compared with subjects living in Western countries.17 Adlercreutz and coworkers found a mean of 276 nmol/L of the isoflavonoid genistein in a sample of Asian men on a regular soy-containing diet.

Genistein and Prostate Cancer A considerable body of evidence has been accumulated that indicates that the Asian soy-rich diet plays an important role in the low incidence of clinically manifested prostate cancer.17 That Asian men who adopt a Western diet and lifestyle lose this protection suggests that nutrition may influence susceptibility for prostate cancer. Genistein inhibits tumor cell growth in experimental models.18 The mechanism of action is complex and includes several cellular pathways. In addition to its antioxidant activity, genistein has been reported to have several biochemical activities such as the inhibition of protein tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR) and src tyrosine kinases,19 the inhibition of topoisomerases,20,21 estrogenic and antiestrogenic activities,20,22 and antiangiogenic activity.23 Genistein arrests the cell cycle and induces apoptosis in gastric cancer, breast cancer, lung cancer, and prostate cancer cells.24-26

Mechanism of Action

Effect of Genistein on Steroid Hormones and Their Receptors The dependence of the prostate on sex hormones for proper growth and development and maintenance of function has become clearer in recent years. Even before the discovery of testosterone, it was well known that a strong dependence existed between the testes and the prostate. After the isolation of testosterone, Huggins and Hodges demonstrated that bilateral orchiectomy was an efficient treatment for advanced prostate cancer, providing the first solid scientific basis for advanced prostate cancer treatment.27 Genistein can interact with the human endocrine system in several ways. One possibility is alteration of hormone concentration. Androgens play an important role in the development and progression of prostate cancer and are the most important risk factor except age.28 Habito et al showed that the replacement of meat protein in the diet with a soybean product did not change total blood concentration of sex hormones, but the mean testosterone-

Figure 1 Molecular Structure and Metabolim of Isoflavonoids Genistein and Daidzein OH

O

OH

OH HO HO

OO

O

OCH3

OH

O

HO

Formononetin

O

OCH3

O Biochanin A OH

OH

O

Genistein

Daidzein HO

OH

Genistin

OH

O

OH

O

OO

HO HO

Daidzin

OH

O HO

O

OH

O

OH

O

HO

O

HO

OH

OH

HO

O Equol

HO OH O-Desmethylangolensin (O-DMA)

OH Tri-OH-Benzene (THB)

HOOC OH

2-(4-hydroxyphenyl) Propanoic Acid

to-estradiol ratio was significantly higher with the meat-containing diet.29 In weaned rats, administration of genistein led to an evident decrease in serum testosterone concentration.30 The biologic functions of steroid hormones are mediated by a family of closely related steroid hormone receptors, with the androgen receptor (AR) mediating the effects of testosterone and related androgens and the estrogen receptor (ER) mediating the effects of estrogens. We, along with other researchers, have been able to show the downregulation of AR protein after the treatment of prostate cancer cells with genistein.31-33 This would result in a modified response of the prostate gland to hormonal stimuli. Estrogen receptor–β (ERβ) seems to be involved in this regulation, as the treatment of LNCaP cells with the antiestrogen ICI 164 384 abrogated AR downregulation.31 Moreover, this prostate cancer cell line is known to express ERβ but not the classical ERα.34 Decreased AR levels result in reduced expression of androgen-stimulated genes. Prostate-specific antigen (PSA), the prototype of an androgen-regulated protein, which is a clinically indispensable parameter used in the diagnosis of prostate cancer as well as for monitoring of treatment response and progression in patients with prostate cancer, is significantly downregulated by genistein treatment.35 Low concentrations of genistein decrease PSA secretion in androgen-sensitive prostate cancer cells, whereas higher doses were needed to significantly inhibit PSA secretion in the androgeninsensitive VeCaP prostate cancer cell line.36,37 This inhibition of PSA synthesis seems to be mediated through androgen-dependent and androgen-independent pathways, as was shown in transfection experiments.37 The expression of another recently identified androgen-responsive gene, prostate androgen–regulated transcript–1, was also reported to be downregulated by genistein.38 Genistein treatment also interferes with key enzymes of steroidogenesis, affecting the level of growth-promoting steroid hormones. Evans and colleagues described the inhibition of 5α-reductase and 17β-hydroxysteroid dehydrogenase by 6 isoflavonoids in human genital skin fibroblast monolayers and homogenates and in benign prostatic hyperplasia tissue homogenates.39 One of the most potent inhibitors of 5α-reduc-

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Molecular Effects of Genistein

Figure 2 Summary of Genistein Effects on Androgen- and/or

Figure 3 Alteration of Cell Cycle Regulation by Genistein

Estrogen-Mediated Tumor Cell Proliferation

Cyclin A

Genistein

CDK2

5a-Reductase

Cyclin cyclin E E

CDK2

PSA

AR AR

–

PART-1

G1 Cyclin D

Cyclin A

st rre p21

+

p27

+

CDC2

st

ERb

Estradiol

Estrone

a

Cyclin cyclin BB

CDK4

Prostate Cancer Cell Growth and Survival

G2

rre

Testosterone

S

a

DHT

17b-HSD

M

CDC2

Inhibition of 5a-reductase and 17b-hydroxysteroid dehydrogenase results in a decrease of dihydrotestosterone and estradiol synthesis, respectively. In this way, genistein can decrease the availability of the 2 most active steroid hormones promoting prostate survival and cell growth. The downregulation of androgen receptor (AR), which seems to occur via estrogen receptor–b (ERb), could result in a modified response to hormonal stimuli and protect the prostate cells from inappropriate proliferative stimuli. Inhibition of expression of androgen-regulated genes such as the PSA gene, which was reported to be mediated through androgen-dependent and androgen-independent pathways, and prostate androgen–regulated transcript–1 (PART-1) can be used as biomarkers for evaluating effects of genistein on the androgen axis.

Treatment of prostate cancer cells with genistein results in downregulation of positive cell cycle regulators, such as cyclin B and CDK4, and upregulation of negative regulators of cell cycle progression, namely p21 and p27, with subsequent inhibition of CDK enzyme activity. These effects cause a G2-to-M phase and/or G1-to-S phase arrest.

tase, the enzyme that catalyzes the irreversible conversion of testosterone to dihydrotestosterone, the most active androgen in the prostate, was genistein, which inhibited the enzyme in a noncompetitive way.39 Moreover, inhibition of 17β hydroxysteroid oxidoreductase leads to a decrease in the availability of the highly active endogenous estrogen.40 Another interference of genistein with steroid hormone action is found at the level of the receptors. Genistein is an estrogen-like compound that can compete with estrogens in binding to the receptors’ ligand-binding site and thus modulating ER function.41 Taken together, these data suggest that genistein is an agent that could modify androgen- and/or estrogen-mediated carcinogenesis (Figure 2).

Cell Cycle Alteration by Genistein

Inhibition of Prostate Cancer Cell Growth by Genistein One of the most important components involved in the control of cell growth and differentiation is the growth factor tyrosine kinases, which are activated by growth factor binding to their cell surface receptors and transmitting the signal to the intracellular signal transduction pathways.42-44 Alteration in the expression of the erbB family members, such as EGFR (ie, ErbB-1), ErbB-2, and ErbB-3, has been demonstrated to be a common event in prostate cancer, thus suggesting these receptor tyrosine kinases as an interesting target for prostate cancer chemoprevention and/or therapy.45,46 Studies on androgen-independent DU-145 prostate cancer cells have identified genistein as a potent inhibitor of ErbB-1, which is constitutively active in this cell line.47 Treatment of prostate cancer cells with genistein resulted also in the inhibition of Shc activation, which is an adaptor protein, resulting in subsequent inhibition of ERK1/2 acti-

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vation.47 These effects of genistein were dose-dependent and without any alteration in protein levels. Dose- and time-dependent inhibition of cell growth by genistein was also found in other prostate cancer cell lines, namely PC3 cells, another AR-negative androgenindependent tumor cell line as well as in the LNCaP cell line, which is AR positive and androgen sensitive.24 Thus, genistein inhibits prostate cancer cell growth independent of AR status.

Abnormalities in the regulation of the cell cycle are among the most common features of transformed cells. Cell cycle progression is an ordered set of events regulated by sequential activation and subsequent inactivation of a series of cyclin-dependent kinases (CDKs). The CDKs, along with the cyclins, are major control switches for the cell cycle, causing the cell to move from G1 to S phase and from G2 to M phase. Several studies indicate that genistein interferes with the regulators of cell cycle progression, causing cell cycle arrest.24,26,48-51 Shen and colleagues suggested that treatment of LNCaP cells with physiologic concentrations of genistein (≤ 20 μmol/L) results in a G1 cell cycle block, probably through upregulation of CDK inhibitors p21WAF1 and p27Kip1, which are negative cell cycle regulators.50 However, an increasing number of studies suggest that genistein is also an inductor of G2/M cell cycle arrest in prostate cancer cell lines.24,26 The inhibition of cell growth was observed with concomitant downregulation of cyclin B1, upregulation of p21 and p27, and a strong decrease in CDK4.24,26 In a recently published work, Oki and colleagues described the induction of growth arrest and DNA damage–inducible gene 45 by genistein as a possible mechanism of genistein contributing to cell cycle arrest.51 Taken together, these data suggest that genistein induces G2/M or G1/S arrest of

Jasmin Bektic et al the cell cycle, depending on cell type, with predominant inhibition of G2-to-M progression in prostate cancer cells. The targets of genistein in cell cycle regulation are summarized in Figure 3.

Genistein and Apoptosis The counterbalance to proliferation in normal cells and tissue is programmed cell death. It allows the organism to tightly control cell numbers and tissue size and to protect itself from cells that threaten homeostasis.52 This process of programmed cell death, which is an active dedicated molecular program, was termed “apoptosis” (from the Greek term for leaves falling off a tree) by Currie and colleagues in 1972.53 Most of the morphologic changes occurring during apoptosis are caused by caspases, members of the cysteine protease family, which are activated specifically in apoptotic cells through cleaving off their prodomains.54 There are 2 families of caspases based on the lengths of their NH2-terminal prodomains. “Initiator” caspases, including caspase 8 and 9 (and also caspases 1, 2, 4, 5, and 10) contain long prodomains,55 which facilitate their interaction with specific adapter proteins. Cytochrome C release from the mitochondria serves as a critical step in the activation of caspase 9. Apoptotic stimuli result in clustering of adapter proteins, bringing initiator caspases in close proximity to one another to promote transcatalytic activation. Initiator caspases then activate various short-prodomain “effector” caspases, including caspases 3, 6, and 7. These in turn cleave structural proteins involved in cell architecture and functional proteins involved in cell cycle regulation and DNA repair.56 The prostate cancer cell growth–inhibiting ability of genistein could also result from induction of apoptosis. Indeed, genistein treatment of PC3 and LNCaP prostate cancer cells resulted in DNA ladder formation and cleavage of poly(ADP-ribose)polymerase,24 which is cleaved by caspase 3 and is used as an indicator of apoptotic cell death.57,58 In PC3 cells, treatment with the caspase 3 inhibitor DEVD-fmk before exposure to genistein significantly inhibited caspase 3 expression and treatment-induced apoptosis, implicating caspase 3 as the main target in genisteininduced apoptosis, at least in this cell line.59 Activation of caspases is regulated directly or indirectly by members of the Bcl-2 and inhibitor of apoptosis (IAP) protein families.60 The IAP protein XIAP directly inhibits the activity of distinct caspases. In contrast, the proapoptotic Bax, Bak, and BH3-only proteins stimulate the release of cytochrome C from mitochondria and the subsequent activation of the apoptosome and caspases, and antiapoptotic Bcl2 family members, such as Bcl2 itself and BclX, inhibit the same. In order to identify the molecular mechanism of genistein-induced apoptosis, its effects on apoptosis-related genes have been investigated. Sarkar and Li found that the expression of Bax is significantly upregulated after genistein treatment.37 The ratio of Bax to Bcl-2, which is considered to be very important to apoptosis, was also significantly increased after genistein treatment, suggesting that upregulation of Bax and downregulation of Bcl-2 may be among the molecular mechanisms by which genistein induces apoptosis. Kazi et al reported that genistein inhibits the proteasomal chymotrypsinlike activity in vitro and in vivo, probably through interaction with the proteasomal β5 subunit.61 This was associated with accu-

mulation of ubiquitinated proteins, among them 3 known proteasome target proteins, ie, the CDK inhibitor p27, inhibitor of nuclear factor–κB (NF-κB), and the proapoptotic protein Bax. Akt (ie, protein kinase B) is a serine/threonine protein kinase that has been implicated in mediating a variety of biologic responses, including inhibiting apoptosis and stimulating survival and cellular growth.62 Its activation occurs via phosphorylation at threonine 308 and serine 473 by PDK1 and PDK2, respectively. When activated, Akt exerts antiapoptotic effects through phosphorylation of substrates such as Bad or caspase 9, which directly regulates the apoptotic machinery or substrates such as the human telomerase reverse transcriptase subunit, forkhead transcription family members, or IκB kinases that indirectly inhibit apoptosis.62 Genistein treatment of PC3 prostate cancer cells showed a decrease of Akt kinase activity and a decrease of Akt protein phosphorylated at Ser473 compared with untreated cells.37 Whereas EGF alone activated Akt kinase, pretreatment of cancer cells with genistein resulted in the abrogation of EGF-induced Akt kinase activation. Likewise, phosphorylation of GSK-3, a downstream substrate of Akt kinase, was inhibited by genistein treatment.37,63 These data suggest genistein as an inhibitor of the Akt survival pathway with consequent initiation of apoptotic cell death. Several studies indicate genistein as a modulator of the transcription factor NF-κB in prostate cancer cell lines. Nuclear factor–κB proteins compose a family of structurally related eukaryotic transcription factors that are involved in the control of a large number of processes, such as immune and inflammatory responses, developmental processes, and cellular growth.64 The activity of NF-κB is tightly regulated by interaction with inhibitory IκB proteins. In most cells, NF-κB is present as a latent, inactive, IκB-bound complex in the cytoplasm. When a cell receives any of a multitude of extracellular signals, such as proinflammatory cytokines and bacterial toxins (eg, LPS and exotoxin B) and a number of viruses/viral products (eg, HIV-1, HTLV-I, hepatitis B virus, Epstein-Barr virus, and herpes simplex) as well as proapoptotic and necrotic stimuli (eg, oxygen free radicals, UV light, and γ-irradiation), NF-κB rapidly enters the nucleus and activates gene expression.65-67 This requires release from IκB, which is a key step for controlling NF-κB activity. There is growing evidence suggesting an important role for NF-κB in the protection against apoptosis.65-67 Genistein modulates NF-κB DNA-binding activity in prostate cancer cell lines.63,68-70 Using an electrophoresis mobility shift assay, genistein was found to significantly inhibit NF-κB DNA binding in hormone-dependent and hormone-independent prostate cancer cells.69 Because the DNA-binding activity of 2 other transcription factors, namely AP-1 and SP-1, was not affected by genistein, investigators suggested an NF-κB–specific inhibition of DNA-binding and transcriptional activity.69 The stimulation of cells with NF-κB inducers leads to phosphorylation and degradation of IκB, allowing the NF-κB translocation to the nucleus, binding to its DNA recognition sites, and regulation of gene transcription. Using immunoprecipitation and Western blot analysis, Davis and coworkers demonstrated that genistein inhibits the phosphorylation of IκB, thus preventing the translocation of NK-κB to the nucleus with subsequent inhibition

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Molecular Effects of Genistein

Figure 4 Schematic Representation of Genistein-Induced Apoptosis in Prostate Cancer Cells

Growth Factors Extracellular

GF

P

Bcl-xL

P

PI3

MEKK

–

Bcl-2

+

Bax

Cytochrome C IKK PDK

PKA

Apaf 1

Bad

proCaspase 9 IkB

p50

p65

Akt –

–

NF-kB

Effector Caspase +

Caspase 3 Caspase 6

p50

p65

Survival

Caspase 7

Apoptosis

Genistein was reported to decrease the expression of the antiapoptotic factor Bcl-2 and to increase the expression of proapoptotic Bax protein. The induction of caspase 3, which belongs to the group of effector caspases, is another molecular mechanism whereby genistein can trigger programmed cell death in prostate cancer cells. In addition, inhibition of Akt and NF-kB activity and their crosstalk contributes to genistein-induced apoptosis.

of cell growth and induction of programmed cell death.69 Of importance for the effects of genistein on NF-κB activity is the crosstalk between the Akt and the NF-κB signaling pathways.71,72 Akt has been shown to enhance the degradation of IκB-inducing NF-κB activity.73 This induction was suggested to be specific for NF-κB, as other inducible transcription factors were not affected by Akt overexpression. Li and Sarkar demonstrated that genistein abrogates the NF-κB activation that follows Akt transfection and/or EGF treatment.63 These data suggest that genistein inhibits NF-κB activation through inhibition of the Akt signaling pathway. The different regulatory pathways involved in the regulation of apoptosis and survival affected by genistein are summarized in Figure 4.

Effect of Genistein on Angiogenesis, Tumor Cell Invasion, and Metastasis The progressive growth and metastasis of tumor cells is dependent on the development of adequate vasculature (ie, angiogenesis) determined by the local balance between positive and negative regulating molecules. Cancer cells exploit this process to recruit their own private blood supply to receive oxygen and nutrients. Significant levels of proangiogenic factors, such as VEGF and interleukin-8, are present in prostate cancer but not in benign prostatic hyperplasia or normal prostate cells.74,75 Another important factor stimulating angiogenesis in prostate cancer is

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transforming growth factor–β (TGF-β).76 Using microarray analysis, TGF-β and several other genes related to angiogenesis have been found to be downregulated after genistein treatment of PC3 prostate cancer cells, suggesting that positive regulators of angiogenesis could be another possible target for this isoflavonoid.77 However, the exact molecular mechanism of the antiangiogenetic potential of genistein still has to be evaluated. Genistein was also reported to alter the metastatic potential of cancer cells. One of the key molecules for cancer metastasis, ErbB2, was found at high levels in different cancers, including a subset of prostate cancer. Transfection studies on breast cancer cells have shown higher metastatic potential of c-erbB-2 overexpressing cells, and this was associated with a higher expression of matrix metalloproteinases (MMPs) 2 and 9.78 Genistein treatment of ErbB-2 transfectants resulted in reduced levels of ErbB-2 and MMPs, suggesting that genistein may reduce metastatic properties of breast cancer cells.79 The corresponding experiments with prostate cancer cells have not been done yet, but MMPs, particularly MMP-9, have already been identified as possible targets for genistein in prostate cancer cells.77

Conclusion Taken together, genistein interacts with several crucial regulatory pathways in prostate cancer cells. Genistein’s reduction of available steroid hormones and/or their receptors, cell cycle arrest, induction of apoptosis, and inhibition of angiogenetic and metastatic potential of tumor cells, in addition to some other effects not mentioned here, are directed to the same purpose: anticarcinogenesis. Although a definitive statement that genistein is a chemopreventive and/or therapeutic agent cannot be definitively made at this time, there is sufficient evidence for the protective effects to warrant further investigation and clinical trials.

References

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