Soy Isoflavone Genistein in Prevention and Treatment of Prostate Cancer

FGE Perabo, EC Von Lo¨w, J Ellinger, A von Ru¨ cker, SC Mu¨ ller and PJ Bastian Department of Urology, University Hospital, Bonn, Germany

Dietary habits and incidence of prostate cancer (PCa) are very different in several parts of the world. Among the differences between Eastern and Western diets is the greater intake of soy in the Eastern cultures. This might be one factor contributing to a lower incidence of PCa in Asian men. Many studies using PCa cells and animal studies of chemical carcinogenesis have shown that a wide range of dietary compounds have cancer chemopreventive potential. Therefore, the interest in nutrition-based approaches for prevention and treatment of PCa is increasing. We reviewed all experimental preclinical in vitro and in vivo data as well as clinical trials performed with soy isoflavone genistein for prevention and treatment of PCa. The preclinical data for genistein presented in this review show a remarkable efficacy against PCa cells in vitro with molecular targets ranging from cell cycle regulation to induction of apoptosis. In addition, seemingly well- conducted animal experiments support the belief that genistein might have a clinical activity in human cancer therapy. However, it is difficult to make definite statements or conclusions on clinical efficacy of genistein because of the great variability and differences of the study designs, small patient numbers, short treatment duration and lack of a standardized drug formulation. Although some results from these genistein studies seem encouraging, reliable or long-term data on tumor recurrence, disease progression and survival are unknown. The presented data potentially allow recommending patients the use of genistein as in soy products in a preventive setting. However, at present there is no convincing clinical proof or evidence that genistein might be useful in PCa therapy. Prostate Cancer and Prostatic Diseases (2008) 11, 6–12; doi:10.1038/sj.pcan.4501000; published online 9 October 2007

Keywords: genistein; isoflavone; prevention; treatment; in vitro; clinical trial

Introduction of fruits and vegetables and the risk of PCa balance between promising or disillusioning results. Many Dietary habits and prostate cancer (PCa) incidence are confounding factors present in clinical studies make very different in several parts of the world. For example, assessment and interpretation of the effects of diet on the incidence of PCa is very low in most of Asia (as low cancer risk difficult. These difficulties are evident in a as 1.0 per 100 000 men). The reason believed for this large study on food consumption and cancer incidence in phenomenon is low animal fat intake together with a diet Europe, the European Prospective Investigation into rich in fiber and soy.1 In contrast, high-fat consumption Cancer and Nutrition, where no association was ob- and a low-phytochemical diet are very common in served between vegetable and fruit intake and the risk of Western countries, with higher incidences of PCa (up to PCa.5,6 Therefore, the large geographical differences 4100 per 100 000 men).2,3 Although there might be some observed are probably not only due to genetic disposi- problematic aspects with cancer registries in Far East tions but relate to complex environmental and socio- countries, there is an additional evidence for the role of cultural (nutrition) factors.7–27 diet in migration studies. It has been shown that In principle, PCa represents an ideal target for individuals with the same genetic background (for nutritional prevention due to its long latency, its high example, Japanese men migrated to the United States) incidence, tumor marker availability (prostate-specific have a risk of PCa comparable to the incidence of PCa of antigen, PSA) and identifiable preneoplastic lesions and the country they live in.4 However, epidemiological risk groups. The interest in nutrition-based approaches studies that investigate the relation between the intake for prevention and treatment of PCa is increasing. However, the interest is most evident in patients already suffering from the disease. Patients use increasingly Correspondence: Dr FGE Perabo, Department of Urology, Bonn complementary and alternative medicines (CAMs) for University, Sigmund-Freud Street 25, Bonn D 53105, Germany. E-mail: [email protected] several reasons: first, in the hope to support the immune Received 3 December 2006; accepted 12 January 2007; published system to fight the cancer in addition to conventional online 9 October 2007 treatment. Second, in the hope to minimize morbidity Genistein in prostate cancer FGE Perabo et al associated with conventional treatment and third in fear downregulated by genistein, whereas p21 mRNA in- 7 of suffering and dying from PCa in the case conventional creased in response to genistein in PCa cells DU-145 and treatment fails.28 CAM is a widely used term comprising LNCaP.42,44 In another study, genistein inhibited endothe- techniques, methods, herbal medicines and nutrition lial cell proliferation and in vitro angiogenesis associated supplements used in addition to mainstream care for with cancer progression at concentrations of 5 and 45 symptom management and improving quality of life 150 mM. Further, it was demonstrated that genistein in cancer patients. Many of these alternative therapies interfered with apoptosis signal transduction by down- comprise unproven methods promoted as treatment or regulating Bcl-XL expression.46 Genistein inhibited the cure with questionable benefit. On the other side, there is nuclear factor-kB (NF-kB) activation via the AKT signal- increasing evidence in the current scientific medical ing pathway and induced apoptosis in androgen- literature on an effectively delivered activity of phy- sensitive PCa cell line LNCaP and the androgen- totherapeutics in modulating cancer cell growth. Phyto- insensitive cell line PC3 in a concentration of 50 mM, chemicals differ from what are traditionally termed whereas no apoptosis was seen in nontumorigenic nutrients because they are not a necessity for normal CRL-2221 human prostate epithelial cells under genistein metabolism nor will their absence result in a deficiency treatment.41,47 The AKT signaling pathway is an im- disease. Many studies using PCa cells and animal studies portant survival pathway in cellular transduction acti- of chemical carcinogenesis have shown that a wide range vated by various growth factors like the epidermal of dietary compounds have cancer chemopreventive growth factor. AKT also regulates the NF-kB activation. potential. This paper critically assesses the preclinical NF-kB activity was completely abrogated in cells and clinical data on one of the most popular phytosub- pretreated with genistein.41,43,47,48 Furthermore, the stances, the soy flavonoid genistein when used in PCa effects of genistein on PC3 cancer cells and experimental prevention and treatment. PC3 bone tumors were evaluated by injecting these cells into human bone fragments previously implanted in immunodeficient (severe combined immunodeficiency, SCID) mice. Genistein significantly inhibited PC3 bone Phytoestrogen genistein tumor growth by regulating the expression of multiple genes involved in the control of cell growth, apoptosis Phytoestrogens are naturally occurring phenolic plant and metastasis both in vitro and in vivo. For example, the compounds classified as flavones, isoflavones, coume- expression of various metalloproteinases (MMPs) in PC3 stans and lignans. They are highly concentrated in soy bone tumors was inhibited by genistein treatment, products (beans, tofu) and plant lignans are found in whereas osteoprotegerin was upregulated. MMP im legumes, whole grain, and various seeds and vegeta- 8,29 munostaining and transfection experiments also demon- bles. Biochemically, phytoestrogens are heterocyclic strated inhibition of MMP-9 expression in PC3 cells phenols with a structural similarity to estrogenic steroids in vitro and PC3 bone tumors in vivo after genistein (mammalian endogenous estrogens) and therefore, treatment.49 these compounds display estrogen-like activity or weak antiestrogen-like properties.29,30 The beneficial effects of a soy diet have been attributed to isoflavones. Genistein, In vivo effects of phytoestrogens the predominant isoflavone in human nutrition is A study with Lobund–Wistar rats that received a high- derived mainly from soybeans but also from other isoflavone diet showed a significant reduction of prostate legumes, including peas, lentils or beans. Quercetin is tumor growth compared to the control group receiving a the main representative of the flavonol class and a low-isoflavone diet.50 Another study with TRAMP mice polyphenolic antioxidant found in a variety of fruits and fed on a genistein-rich diet also found reduction of tumor vegetables. It is highly concentrated in onions, broccoli, incidence.51 Additionally, genistein lowered androgen apples, grapes (red wine) and in soybeans, and is also a and estrogen-receptor expression in the rat prostate, component of gingko biloba. shown by a study of a diet with 250–1000 mg genistein per kg fed to male Sprague–Dawley rats.52,53 These, and other trials with animal models provide promising In vitro effects of phytoestrogens data for treatment of PCa with isoflavonoids.54–57 A Physiological concentrations of the soy-derived isofla- compilation of the preclinical in vitro and in vivo data is vone genistein were shown to downregulate the andro- shown in Table 1. gen receptor of PCa cells via the estrogen receptor b, resulting in a modified response to hormonal stimuli.31 They also inhibit several steroid-metabolizing enzymes Clinical data on genistein such as 5-a-reductase or aromatase.32,33 It has been Although there are plenty of experimental data available, postulated that these activities may be protective for PCa large epidemiological trials to underline the antitumoral by creating a more favorable hormonal milieu. Isofla- effect of isoflavones are rare. The first prospective cohort vones have been well analyzed in human PCa cells over study was conducted in 1994 and showed that flavonoid the past years. Genistein inhibited the growth and intake was not associated with mortality from cancer.16 induced apoptosis in LNCaP, DU-145 and PC3 prostatic This was confirmed in a cross-national study of seven 34–42 cancer cells in a concentration p20 mM. Genistein countries with 16 cohorts. A positive effect on coronary blocked the cell cycle progression at G1, inhibited PSA heart disease might be attributed to flavonoid intake but expression and modulated cell cycle gene regula- not cancer mortality.60 However, another cross-national tion.37,38,40,43 Expression of telomerase reverse transcrip- study from 42 countries found that soy products were tase, c-myc mRNA and the MDM2 oncogene were significantly protective on PCa mortality (P ¼ 0.0001)

Prostate Cancer and Prostatic Diseases Genistein in prostate cancer FGE Perabo et al 8 Table 1 Summary of molecular targets affected by genistein in vitro and in vivo in androgen-insensitive (PC3, DU-145) and androgen- sensitive (LNCaP, 267B-1, BRFF-41T, PNT-1, PNT-2 and SKRC-1) prostatic cell lines Target Cell culture system/animals

Genistein in vitro Inhibition of tumor cell growth LNCaP,36,39,54 PC3,35,36 DU-145, PNT-1, PNT-2,35 LNCaP in SCID mice54,55 Induction of apoptosis LNCaP,38,39 PC3,38 LNCaP, PC3, DU-145 in SCID mice54 Inhibition of PSA expression LNCaP,37,39,58 VeCaP58 Suppression of DNA synthesis LNCaP39 Downregulation of cyclin-B LNCaP, PC338 Upregulation of p21 WAF1 protein LNCaP,37,38 PC338 Upregulation of p27KIP1 protein LNCaP37 Inhibition of NF-kB activation LNCaP, PC3,47 PC341 Modulation of AKT signaling pathway PC341 37 Induction of G1 cell cycle block LNCaP 36 Induction of G2/M cell cycle arrest LNCaP, PC3 Inhibition of mitogenic signaling pathways DU-14557 Reduction of CYP27B1 and CYP24 protein levels DU-14559

Genistein in vivo Induction of DNA fragmentation, reduction of tumor vessel LNCaP, PC3, DU-145 in SCID mice54 density, reduction of serum IGF level Inhibition of tumor growth, apoptosis Nude mice/LNCaP56 Suppression of tumor growth Lobund–Wistar rats50 Development and function of the rat dorsolateral prostate Sprague–Dawley rats53 Androgen and estrogen receptor expression Sprague–Dawley rats52

Abbreviations: IGF, insulin-like growth factor; NF-kB, nuclear factor-kB; PSA, prostate-specific antigen; SCID, severe combined immunodeficiency.

with an effect size per kilocalorie at least four times as (n ¼ 6), off-cycle during hormonal therapy (intermittent large as that of any other dietary factor.61 A substantial hormones; n ¼ 14) or active surveillance (n ¼ 16). The review of studies that have assessed the direct relation primary end point for the trial was a 50% reduction in between the individual dietary intake of soy products the PSA level at 6 months compared with before and the risk of PCa was done by Ganry in 2005. Ganry treatment. Fifty-two patients were available for evalua- analyzed epidemiological studies providing data on (1) tion at 6 months. One of 52 patients had a more than 50% dietary soy intake or flavonoids intake, (2) urinary reduction in the PSA level, additional 7 patients had PSA excretion of isoflavones or lignans or (3) blood measure- reductions that were less than 50%. All eight patients ments of isoflavones or lignans. Soy was used as a with lower PSA levels at 6 months were in the active marker for isoflavone intake. Overall, the results of these surveillance (watchful waiting) treatment subgroup. studies did not show protective effects. Only four of Repeated measure regression models allowing for these studies were prospective, and none of them found correlation between initial levels and change also statistically significant PCa reductions.11 indicated a decline in PSA in this group compared with However, there are some phase I and II trials other groups: 0 of 52 had a complete response, 9 (17%) evaluating efficacy and safety of genistein in patients had a partial response, 8 (15%) had stable disease and 35 with PCa. In a nonrandomized, nonblinded trial of 38 (67%) had disease progression. Taken together, genistein patients (20 with clinically significant PCa and 18 may hold some promise in PCa treatment but more controls), a daily intake of 160 mg isoflavones extract study is warranted.64 A summary of clinical trails with until radical prostatectomy led to significantly higher genistein is depicted in Table 2. apoptosis of tumor cells (P ¼ 0.0018). No adverse events were reported, the median treatment time was 20 days.62 With the objective to assess the potential genotoxicity of a purified unconjugated isoflavone mix, Miltyk et al.63 Discussion observed 20 PCa patients treated with 300 mg genistein per day for 28 days and with 600 mg per day for 56 days To establish the influence of a nutritional compound as but could not find any significant genetic damage. A genistein on cancer genesis, promotion or progression study by De Vere White et al.64 tried to determine carefully designed, larger-scale, prospective randomized whether supplemental amounts of soy isoflavone (genis- trials should further support the epidemiological and tein-rich extract) would lower PSA levels more than 50% experimental data. Numerous case–control studies, but in patients with PCa. A total of 62 men with histologi- unfortunately only a few prospective randomized cally proven PCa who had two consecutive elevated PSA clinical trials have analyzed the influence of diet on readings were accrued into an open-label pilot study. PCa. Evidence-based data are needed to definitively Patients took capsules containing the genistein-rich prove the efficacy of genistein or other phytochemicals extract three times daily by mouth. The subjects for PCa because of the growing interest in and use of were in one of five groups: after radical retropubic herbal remedies as a persistent trend of our present-day prostatectomy (n ¼ 9), after radiotherapy (n ¼ 17), after health care. The summary of preclinical data for both radical retropubic prostatectomy and radiotherapy genistein presented in this review shows a remarkable

Prostate Cancer and Prostatic Diseases Genistein in prostate cancer FGE Perabo et al 9 Table 2 Compilation of recent clinical studies on soy isoflavones (genistein, daidzein) for PCa Substance/preparation/ Patient Efficacy/response Toxicity Clinical Reference scheduling characteristics phase

Normal ‘Western’ diet 63 pts with BPH Genistein plasma levels were greater in Not noted Phase I 8 31 pts with PCa group A (small prostate) than in group B (large prostate) (P ¼ 0.023) and in group C (PCa) similar to the entire BPH group (P ¼ 0.34); PSA level in group C was greater than in BPH groups (P ¼ 0.004); BPH volumes were greater in groups A+B than in C (P ¼ 0.037)

Soy beverages twice 34 elderly men with ISP+ and ISPÀ increased serum No systemic or local Phase I 65 daily for 6 weeks period elevated PSA concentrations and urinary output of toxicity noted ISP+(42 mg genistein the isoflavones and their metabolites, plus 27 mg daidzein) IPS+ decreased serum cholesterol, no ISPÀ (2.1 mg genistein effect on serum PSA or p105erB-2 and 1.3 mg daidzein) protooncogene

Genistein preparation 20 pts with PCa 6 A single elevated micronucleus No systemic or local Phase I 63 (43% genistein, 21% controls frequency (MF) was found in pt. No toxicity noted daidzein, 3% glycitin) genistein-induced rearrangements of 300 mg dayÀ1 for 28 days the MLL gene were detected. Total and then with 600 mg genistein never exceeded a peak dayÀ1 for another concentration of 27.1 mmol lÀ1, 56 days unconjugated genistein never exceeded a peak concentration of 0.32 mmol lÀ1

Soy formulation 20 pts with PCa Serum DHT decreased by 31.7% Mild estrogenic effects Phase I 66 300 or 600 mg genistein (P ¼ 0.0004), genistein and daidzein like breast changes, and were rapidly cleared from plasma frequency of hot 150 or 300 mg daidzein and excreted in urine. flushes for 84 days

Purified soy isoflavones 30 healthy men Pharmacokinetic analyses in 24 h after Well tolerated, no Phase I 67 A (90% genistein, 10% 5 dose groups— single-dose administration on mean physical changes, daidzein, 1% glycitein) each with 6 subjects elimination half-lives for free genistein elevation of B (43% genistein, 21% were 3.2 and 4.2 h for free daidzein with lipoprotein lipase and daidzein, 2% glycitein) both formulations hypophosphatemia

68 Unconjugated soy 11 pts with PCa, Maximal plasma concentrations (Cmax One of 13 patients Phase I isoflavones 2 pts with colon Ca ranged between 4.3 and 16.3 mM for total treated developed a PTI G-2535 (43% genistein and 0.066 and 0.17 mM for free treatment-related genistein, 21% daidzein, genistein). For PTI G-2535 and PTI rash. No other 3% glycitin) G-4660, half-lives were 15.03 and toxicities were PTI-G4660 (90% 22.41 h, respectively, and volume of observed genistein, 9% daidzein, distribution was 189.9 and 653.8 l, 1% glycitin) respectively, there was a trend toward Cohorts of four patients higher AUC for PTI G-2535 (P ¼ 0.07 at received single doses of the 8 mg kgÀ1 dose). Treatment-related 2, 4 or 8 mg kgÀ1 orally, increases in tyrosine phosphorylation each dose was separated were observed in PBMC. Plasma by 1 week concentrations of genistein are achieved that have been associated with antimetastatic activity in vitro

Diet rich in soy 28 pts with PCa Statistically significant differences were No systemic or local Phase I/II 69 and linseed soy group 1I (n ¼ 8), detected between the soy group and the toxicity noted Soy group (high soy and linseed wheat group for the % of change in total phytoestrogen) group 2 (n ¼ 10), PSA (12.7 vs 40%, P ¼ 0.02) and the Soy and linseed group wheat group 3 percentage of change in free/total PSA (high phytoestrogen) (n ¼ 8) ratio (27.4 vs 15.6%, P ¼ 0.01) and Wheat group (low between the soy group and the soy/ phytoestrogen) linseed group for the % of change in free androgen index (16.4 vs 15.5%, P ¼ 0.04) and the % of change in free/total PSA ratio (27.4 vs 10%, P ¼ 0.007)

Dietary isoflavones 38 pts before Apoptosis in radical prostatectomy No systemic toxicity Phase II 62 160 mg dayÀ1 containing therapy of PCa 18 specimens from treated patients was noted genistein, daidzein, controls significantly higher than in control formononetin, biochanin subjects (P ¼ 0.0018), specifically in A for 6 weeks regions of low- to moderate-grade cancer (Gleason grade 1–3).

Prostate Cancer and Prostatic Diseases Genistein in prostate cancer FGE Perabo et al 10 Table 2 Continued

Substance/preparation/ Patient Efficacy/response Toxicity Clinical Reference scheduling characteristics phase

Isoflavones 41 pts in 3 groups Median follow-up was 5.5 months; 39 No systemic toxicity Phase II 70 2 Â 100 mg soy dayÀ1 Group I: watchful pts were analyzed, no complete or noted for 3–6 months waiting with rising partial response, stable disease (PSA) PSA (n ¼ 4), was 83% in group II and 35% in group Group II: increasing III, no changes in serum levels of PSA with local testosterone, IGF-1, IGFBP-3 or therapy (n ¼ 18); 5-OHmdU Group III: with hormone therapy (n ¼ 19) Genistein-rich extract 62 pts with PCa CR was 0%, 9 pts (17%) had a PR, 8 3 pts discontinued Phase II 64 capsules (450 mg dayÀ1) who had two (15%) had SD and 35 (67%) had disease because of adverse 3 capsules for 6 month consecutive progression. The total testosterone level events (diarrhea) and elevated PSA was lowered in one of the patients 7 because of personal responding, but it was higher in choice five others Soy isoflavone (60 mg 76 pts with PCa 59 pts completed 12 weeks, serum free No systemic or local Phase II 71 dayÀ1) or placebo testosterone was reduced or unchanged toxicity noted for 12 weeks of 61% in the isoflavone group vs 31% in the placebo group, serum total PSA decreased or remained unchanged in 69% in the isoflavone group vs 55% in the placebo group

Abbreviations: AUC, area under the concentration curve; BPH, benign prostatic hyperplasia; CR, complete response; DHT, 5-a-dihydrotestosterone; MLL, myeloid/lymphoid or mixed lineage leukemia; PBMC, peripheral blood mononuclear cells; PCa, prostate cancer; PSA, prostate-specific antigen; PR, partial response; pts, patients; SD, stable disease.

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