Research article
Synergistic chemoprotective mechanisms of dietary phytoestrogens in a select combination against prostate cancer☆
Introduction
Prostate cancer (CaP) is the most frequently diagnosed malignancy in men, and though only a small proportion of low-risk CaPs progress to life-threatening disease during the lifetime of the patient, it is still the second most common cause of cancer death [1]. It has been established that the incidence of clinically significant CaP is much lower in parts of world where people eat predominantly a plant based diet [2], and therefore the interest in phytoprevention and phytotherapy of CaP has increased considerably [3]. This is also important from the point of view that many men with low-risk CaP choose comprehensive life-style changes (including plant-based nutrition) over surgery/radiotherapy for disease management [4]. Invasive prostate carcinomas become androgen insensitive due to mutation of androgen receptors [5], [6]; nevertheless their estrogen sensitiveness increases as a result of an increased expression of estrogen receptors (ERs) [7]. Phytoestrogens, because of their structural similarity to estradiol, bind to these ERs [8], especially ER-β, which is anti-proliferative and proapoptotic [9] and is expressed in plenty in normal prostate as well as CaP [7], [10]. Studies have shown that selective ER modulators (including phytoestrogens) inhibit CaP cell proliferation in vitro [11], [12] as well as in vivo [13]. Besides estrogen agonistic/antagonistic activities, phytoestrogens also exert a number of other effects that beneficially modulate several aspects of tumor growth [14]. However, a major constraint in their use as effective chemoprotectants is poor bioavailability and toxicity at efficacious doses. Vegetarian diets contain combinations of different phytochemicals that may act synergistically to modulate the aberrant signaling pathways of cancer cells resulting in chemoprotective effect at physiological concentrations, as reflected in epidemiological studies [15]. Hence, simultaneous administration of dietary phytochemicals with different chemopreventive mechanisms may provide additive or synergistic activity at lower doses of the individual agents, resulting in improved efficacy and reduced toxicity [16]. We selected some phytoestrogens on the basis of their diverse anti-cancer mechanisms (as reported in literature), not necessarily requiring their binding to the ER (which is a common property of all phytoestrogens). This was done to selectively combine phytoestrogens with different anticancer mechanisms together, so as to get a combination effect. Several combinations were tried, and we identified a combination of genistein (G), biochanin A (B) and quercetin (Q) that was most effective in inhibiting prostate cancer cell proliferation, in vitro. G is a potent inhibitor of tyrosine kinases [17], B is a powerful agonist of the human aryl-hydrocarbon (ArH) receptor [18] and Q is strongly anti-inflammatory [19], [20]. Inhibition of tyrosine kinases by G and activation of ArH receptor by B inhibits prostate carcinogenesis [21], [22] while Q mediates suppression of inflammation, which plays a crucial role in pathogenesis of CaP [23]. A rational approach to CaP prevention/treatment using dietary phytoestrogens could include a combination of potent ArH receptor agonism, tyrosine-kinase inhibition and anti-inflammatory activity, and therefore, we studied the combined and individual effects of B, G and Q on prostate cancer cell lines in vitro. To the best of our knowledge, this is the first experimental evidence on a multiple phytoestrogen combination exhibiting significantly enhanced efficacy against prostate cancer cells than three times higher concentration of any individual component.
Section snippets
Cell culture
Androgen-responsive prostate cancer cells (LNCaP), DU-145 and PC-3 prostate cancer cells were procured from the American Type Culture Collection (ATCC, Manassa, VA, USA). PC-3 and DU-145 cells were grown in Dulbecco's modified eagle medium/Ham's F-12 (1:1; without phenol red; Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (charcoal stripped, Life Technologies), 100 U/ml penicillin G sodium and 100 μg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) in an atmosphere of
Dose dependent inhibition of prostate cancer cell proliferation by phytoestrogens at noncytotoxic concentrations
Initially, the dose-dependent growth inhibitory potential of G, Q and B against prostate cancer cells was evaluated by MTT assay. All the three exhibited almost equipotent activity and caused a dose-dependent inhibition of prostate cancer cell proliferation in vitro (data not included). Cytotoxicity of these phytoestrogens was determined against a nonprostate, noncancer Vero (monkey kidney epithelial) cell line by employing the MTT assay. The compounds were found to be safe up to 50 μM as the
Discussion
In LNCaP, a direct association between the expression of androgen receptor (AR) and ER-β has been reported [24], and therefore, ER-β agonists like selective ER modulators and phytoestrogens act quite substantially through AR by down-regulating its expression [25] and, consequently, the expression of AR dependent genes [26]. Prostate cancer in its initial stages is androgen responsive and regresses by androgen ablation. However, it generally returns in a more invasive and hostile form that is
Acknowledgments
Authors acknowledge the active participation of Miss Cho Alakammai in this work as a research apprentice. R.K., V.V. and A.J. received research fellowships from the Indian Council of Medical Research, New Delhi, India. This study was supported by a grant from the Ministry of Health and Family Welfare, Government of India.
References (59)
- et al.
Androgen receptor alterations in prostate cancer relapsed during a combined androgen blockade by orchiectomy and bicalutamide
Lab Invest
(2001) - et al.
Metastases of prostate cancer express estrogen receptor-beta
Urology
(2004) - et al.
Comparative studies of the estrogen receptors beta and alpha and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma
Am J Pathol
(2001) - et al.
clinical trial to test the efficacy and safety of Toremifene in men with high-grade prostatic intraepithelial neoplasia
Clin Prostate Cancer
(2003) - et al.
Kaempferol and quercetin stimulate granulocyte-macrophage colony-stimulating factor secretion in human prostate cancer cells
Mol Cell Endocrinol
(2008) - et al.
Progress in cancer chemoprevention: development of diet-derived chemopreventive agents
J Nutr
(2000) - et al.
Molecular effects of the isoflavonoid genistein in prostate cancer
Clin Prostate Cancer
(2005) - et al.
Red clover isoflavones biochanin A and formononetin are potent ligands of the human aryl hydrocarbon receptor
J Steroid Biochem Mol Biol
(2008) - et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J Biol Chem
(1987) - et al.
The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate
J Biol Chem
(1998)
PI3K: downstream AKTion blocks apoptosis
Cell
Activation of extracellular signal-regulated kinase in human prostate cancer
J Urol
bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer
Urology
Cancer statistics, 2008
CA Cancer J Clin
Comparative epidemiology of cancers of the colon, rectum, prostate and breast in Shanghai, China versus the United States
Int J Epidemiol
Soy isoflavone genistein in prevention and treatment of prostate cancer
Prostate Cancer Prostatic Dis
Changes in prostate gene expression in men undergoing an intensive nutrition and lifestyle intervention
Proc Natl Acad Sci U S A
Androgen receptor mutations in prostate cancer
Cancer Res
Soy isoflavones — benefits and risks from nature's selective estrogen receptor modulators (SERMs)
J Am Coll Nutr
Estrogen receptor beta regulates epithelial cellular differentiation in the mouse ventral prostate
Proc Natl Acad Sci U S A
Raloxifene, a mixed estrogen agonist/antagonist, induces apoptosis in androgen-independent human prostate cancer cell lines
Cancer Res
Involvement of the estrogen receptor beta in genistein-induced expression of p21(waf1/cip1) in PC-3 prostate cancer cells
Anticancer Res
Innovative agents in cancer prevention
Recent Results Cancer Res
In vivo quercitrin anti-inflammatory effect involves release of quercetin, which inhibits inflammation through down-regulation of the NF-kappaB pathway
Eur J Immunol
Quercetin inhibits IL-1 beta-induced ICAM-1 expression in pulmonary epithelial cell line A549 through the MAPK pathways
Mol Biol Rep
The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase
Apmis
The aryl hydrocarbon receptor inhibits prostate carcinogenesis in TRAMP mice
Carcinogenesis
The role of inflammation in the pathogenesis of prostate cancer
J Urol
Androgen receptor or estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation and PSA production in human prostate cancer cells
Prostate
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Supported by a grant from the Ministry of Health and Family Welfare, Government of India; CDRI communication No. 7796.