Prostate cancer is the second leading cause of cancer-related deaths in North America. Since the introduction of androgen ablation therapy by Huggins and Hodges in the 1940's, hormone manipulation has been used as the main therapy for advanced PCa 
. Despite initial tumor regression following hormonal therapy, some remaining PCa cells may survive this therapy, acquire androgen independency and become hormone refractory 
. Once the disease progresses to the hormone-refractory state, there is no effective treatment currently available 
. Therefore, a considerable amount of effort has been invested into strategies to treat advanced PCa. Previous in vitro
studies have suggested that genistein has chemotherapeutic potential on hormone-dependent cancer cell lines. However, genistein's in vivo
actions have recently been challenged by contradictory reports 
. For example, one study reported that genistein inhibited PC3 bone tumor growth in SCID mice 
, and another showed that genistein decreased lung metastasis in androgen receptor-negative PC3-M implanted mice, which were fed genistein-enriched chow 
. In contrast, Raffoul et al.
found that genistein ingestion lead to an increase in lymph node metastasis in their PC3 implanted animal model 
. In 2009, Touny and Banerjee demonstrated biphasic effects of genistein using the TRAMP mouse (transgenic adenocarcinoma mouse prostate) model. Genistein inhibited poorly differentiated PCa in these mice when incorporated in their diet before tumor initiation (ie, when the mice were fed genistein-diet between 4~12 weeks of age). However, if the genistein-diet was given to the older TRAMP mice at ages of 12~20 weeks, when prostatic intraepithelial neoplasia (PIN) was already present, it promoted PCa progression and induced lymph node metastasis 
. From such data, it can be implied that life-time moderate consumption of (or early exposure to) genistein is important in prevention of the disease, but that genistein may not exhibit chemotherapeutic effects in vivo
once PCa has already been established or progressed to an advanced stage.
To resolve the controversy of genistein's effects in vivo
, we developed a clinically relevant xenograft model that has been generated from a patient prostatectomy specimen. The resultant patient-derived prostate tumor line used in our study, LTL163a, has been passaged for only 10 generations in immune-deficient mice and has maintained the original histopathological and genotypical characteristics of the original clinical sample 
. Our data show that both low and high dose genistein treatments promote metastasis in this advanced human PCa transplant line. Although the tumor size was not significantly different between control and genistein-treated groups after 3 weeks of treatment, metastatic incidence was greater in genistein-treated vs untreated controls. The metastatic progression observed in our model was characterized by increased cell proliferation and decreased apoptosis. One of the possibilities for the non-significant difference in tumor size between groups while genistein treatment increased proliferation is that all tumor grafts regardless of treatment had reached the maximum outward growth within the renal capsule, after which point started to invade inwardly into the kidney, then to LN and to secondary organs.
It is intriguing to note that genistein exhibits biphasic effects in hormone-dependent cancer cell lines, depending on the dosage 
. Studies with breast cancer cell lines have shown that low-dose genistein (0.1–25 µM) stimulated growth of estrogen-dependent cells, while high-dose genistein (50–100 µM) inhibited proliferation 
. Wang et al.
also demonstrated a similar biphasic effect of genistein in a non-cancerous prostate epithelial cell line (RWPE cells) 
. In our study, however, we did not observe biphasic effects of genistein with the dose range of 80 mg/kg/day and 400 mg/kg/day. In order for genistein to exhibit inhibitory effects on advanced PCa in vivo
, it may require higher dose administration.
Genistein has been shown to modulate activities of PTKs 
. PTKs play a central role in regulating cellular functions such as proliferation, apoptosis, differentiation and cell survival 
. PTK activity is, thus, important in the process of carcinogenesis and metastasis. EGFR is a membrane-associated tyrosine kinase, and its activity is regulated by ligand-binding and by interactions with EGFR family member receptors 
. Once this molecule is phosphorylated, the activated signal is passed down through signaling cascades, activating numerous downstream molecules, which ultimately affects cell division, proliferation and cell migration 
. Abnormal signaling in EGFR-related pathways leads to uncontrolled cell growth and has been reported in many solid tumors such as breast, colorectal and head and neck and pancreatic cancers 
. Src is a member of a non-receptor tyrosine kinase located in cytoplasm and is one of the downstream molecules of EGFR 
. Dysregulation of Src has been linked to oncogenesis process for many years 
, and it is also known to play a role in metastasis by modulating cell motility and invasive abilities in skin, breast and colon cancers 
. Our data show that genistein increases phosphorylation levels of EGFR and Src in an advanced human PCa, which are linked to enhanced cell proliferation and decreased apoptosis. Although previous in vitro
studies demonstrate that genistein inhibits some PTK activities and thus impedes cell growth in cultured cells, our study shows that it increases phosphorylation of EGFR and Src in vivo
, which is in agreement with the results from a phase I clinical trial that reported a ‘surprising’ increase in protein tyrosine kinase phosphorylation after oral administration of genistein 
. As demonstrated here, it is important to note the difference between studies utilizing cultured cells versus in vivo
studies that more closely mimic clinical cancer 
. What contributes to this difference may lie in the dynamic tissue interactions that exist in the tumor microenvironment unique to in vivo
models. For example, growth factors released by the stroma or systemic hormonal influence on cells may affect their biology in ways that cannot be replicated by isolated cancer cells in vitro
Because of genistein's preferential binding to ERβ 
and the high and exclusive expression of ERβ in our tumor line (no ERα expression; Figure S2
), it can be hypothesized that genistein's tumor stimulatory effects observed in this study may be mediated via ERβ activation. It has been shown previously that non-genomic signaling of ERs can activate PTKs 
. In a non-tumorigenic prostate epithelial cell line (RWPE-1), which predominantly expresses ERβ, Wang et al.
showed that genistein treatment at low concentrations (0–12.5 µmol/L) increased cell proliferation and activity of extracellular signal regulated kinase (ERK) 1/2. They have also shown that anti-estrogen treatment with ICI 182, 780 inhibited genistein-induced cell proliferation and activities of ERK1/2 
. Another study by Migliaccio et al.
demonstrated that ligand-activated-ERβ/androgen receptor (AR) complex associated with Src, which then activated the Src/Raf-1/Erk-2 pathway, stimulating cell proliferation in LNCap cells 
. As demonstrated by the LNCap and RWPE studies 
, genistein-activated ERβ may stimulate the EGFR/Src signaling pathway, which leads to increased proliferation, reduced apoptosis and tumor progression in our model. More studies are needed to determine how estrogenic activities of genistein affect tumor growth in vivo
In summary, this study has demonstrated that genistein promotes metastatic activity in advanced PCa. It is possible that genistein has heterogeneous actions through which promotes cancer growth and progression in certain subtypes of cancers while inhibiting other tumors due to differential ERβ expressions among patients. Some PCa cells may have higher expression of ERβ than other cancer cells, favoring the progression of the disease. Future studies focusing on genistein's estrogenicity and its interaction with ERβ will help identify the subtypes of cancers whose growth is promoted or suppressed by genistein, facilitating rational planning of future clinical trials and minimizing the impact of genistein's adverse effects on cancer patients. A better understanding of genistein's cancer promoting actions may lead to the discovery of new therapeutic avenues.