| Background and Objectives: As the most frequently diagnosed cancer in men, prostate cancer is one of the leading malignancies in the male genitourinary system, pathologically including prostate epithelial malignancies, ductal adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, and urothelial carcinoma. According to 2012 statistics, there are 1,111,700 cases of new patients in the world, with 758,700 cases of new patients being diagnosed in developed countries and 353,000 cases of new patients in developing countries. The incidence rates vary more than 25-fold country to country, with the highest rates being recorded primarily in the developed countries of Oceania, Europe, and North America. Prostate cancer is the sixth leading cause of cancer death in men. 307,500 people died of prostate cancer in 2012, accounting for 3.75% of the total cancer in men. There were 142,000 deaths in developed countries and 165,500 deaths in developing countries. The mortality in developing countries was significantly higher than that in developed countries.There are also remarkable racial differences in incidence rate and mortality, e.g., blacks have much greater incidence rate and death rate than whites and Asians. Although the incidence rate of prostate cancer in South and East Asia are relatively lower than that in European and American countries, the trends of increasing incidence and poor prognosis are still of concern. For instance, compared with developed countries, the incidence of prostate cancer is increasing every year in Asia and Eastern Europe regions. Additionally, the incidence rates of prostate hyperplasia in Japan and China are close to that in Western countries.Gleason scoring system, ranging from 2-10, is used for clinical classification of prostate cancer, providing a basis for selection of different treatment modalities, such as surgery, radiation therapy, focused ultrasound, chemotherapy, and hormonal therapy or the combination of these approaches. The clinical efficacy of these treatments depends on the disease status and the intrinsic effectiveness of the therapy. In general, early detection is the key to a successful treatment and cure. Unfortunately, patients often develop into hormonal resistant prostate cancer(HRPC) after endocrine therapy, resulting in disease progression and decreased quality of life among the patients. Therefore, it is an urgent need for effective interventions at early stage prostate cancer and novel therapeutic approach to HCRP, in order to improve the prognosis and the quality of life of patients with prostate cancer.In the search for novel effective and safe interventions for prostate cancer, natural products and derivatives become an attractive source for novel preventive and therapeutic agents. Epidemiological studies have indicated that the low incidence of prostate cancer in Asian men may be related to a high consumption of soy foods. The soy isoflavones in soy foods have been shown to exert anti-tumor effects in vitro and in vivo. Among those isoflavones, daidzein has been shown to have anticancer activity. It is mainly present in soybeans and their products. Experimental data indicate that daidzein is metabolized by colonic bacteria to form equol via an intermediate dihydrodaidzein. In comparison with other isoflavones, equol is unique in chemical structure, having a chiral carbon atom at position C-3 of the furan ring. Equol has two distinct enantiomeric forms, S-equol and R-equol. It has recently been shown that S-equol enantiomer is almost an exclusively daidzein metabolite resultant from human intestinal bacteria. However, there is a remarkable racial difference in the production of the bacterial metabolites; approximately 30% of Western adults and 60% of Asian adults have the ability to produce equol from Daidzein following a soy challenge.Although the molecular mechanisms for equol’s biological activities have not been fully understood, it has been indicated that is equol is a selective estrogen receptor(ER) modulator, having a selective affinity for ER-β. S-equol binds to ER-β with approximately 20% as much affinity as 17b-oestradiol and has a much stronger affinity for ER-β compared to R-equol. Equol also has stronger in vitro antioxidant activity than other isoflavones. There are increasing evidences supporting the notion that equol has other molecular targets in relation to its anticancer activity. For instance, equol inhibits MMP2 and MMP9, which is responsible for its inhibitory effects on prostate cancer metastasis observed in in vitro models. Equol can also activate extrinsic apoptosis pathway through Caspase8 and promote cytochromec release to activate mitochondria-mediated apoptosis.The present study was designed to identify and valuated novel molecular targets for Equol, including several signaling pathways such as Forkhead box O3 and MDM2 pathways. Forkhead box O3, also known as FOXO3 or FOXO3a, is a human protein encoded by the FOXO3 gene. FOXO3 belongs to the O subclass of the forkhead family of transcription factors which are characterized by a distinct fork head DNA-binding domain.FOXO3 a functions as tumor suppressor by up-regulating genes involved in the control of the cell cycle(p27Kip1 and p21Cip1) or in the induction of apoptosis(Fas ligand [Fas-L] and Bim).The transcription factors share the ability to be inhibited and transported out to the cytoplasm on phosphorylation at three conserved sites(T32, S253, and S315) by proteins such as AKT or Protein kinase B(PKB) in the PI3 K signaling pathway. There are many FOX3 interactive molecules involved in the regulation of its expression and function. In addition to AKT, other kinases, such as the serum and glucocorticoidinducible kinase(SGK), casein kinase 1(CK1), dual tyrosine phosphorylated regulated kinase 1(DYRK1), extra-signal-regulated kinases 1 and 2(ERK1/2), and IκB Kinase β(IKK) can also phosphorylate FOXOs and therefore regulate their subcellular localization. MDM2, an E3 ubiquitin ligase, has been reported to mediate FOXO3 a ubiquitination and degradation after ERK-dependent phosphorylations. AKT/PKB is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. AKT possesses a protein domain known as a PH domain, which binds to phosphoinositides with high affinity. Once correctly positioned at the membrane via binding of PIP3, AKT can be phosphorylated by its activating kinases, phosphoinositide dependent kinase 1(PDPK1 at threonine 308) and the mammalian target of rapamycin complex 2(m TORC2 at serine 473). After phosphorylated, AKT can regulate phosphorylation of its downstream proteins. The MDM2 oncogene is a multi-facet molecule that has both p53-dependent and p53-independent activity and plays an important role in human carcinogenesis and cancer development and progression. It has been validated as an anticancer target.The present study aimed at testing the hypothesis that AKT/FOXO3 pathway is critical for S-equol to exert its anticancer activity. To that end, in vitro and in vivo prostate cancer models were used.Methods: The MTT assay was used to detect the inhibitory efficacy of S-equol in prostate cancer cells in comparison with normal prostate epithelial cells to demonstrate its activity and specificity. Flow cytometric analysis was used to detect the effects of S-equol on cell cycle and apoptosis in prostate cancer cells. Immunofluorescence assay was used to detect the effects of S-equol on FOXO3 protein nuclear localization. Western blotting analysis was used to determine the expression levels and the phosphorylation levels of FOXO3 a and AKT and the expression levels of p21, p27, FasL, and Bim. The nude mouse xenograft tumor model of human prostate cancer PC3 cells was used to demonstrate the in vivo efficacy of S-equol as an anti-prostate cancer agent.Results: S-equol inhibited cell survival in three prostate cancer cell lines, Ln CaP, DU145, and PC3, with half-maximal inhibitory concentrations(IC50) values ranging from119.2μM to 169.13μM. S-equol had minimal effect on the survival of RWPE-1, a human non-malignant prostate epithelial cell line. After S-equol treatment, the three prostate cancer cell lines exhibited a significant increase in apoptosis(p<0.05) in a concentration-dependent manner; compared with the DMSO control cells, following exposure to 200 μM of S-equol, the apoptotic indices were increased by 1.88,1.54, and 3.3-fold in Ln Ca P, DU145, and PC3 cells, separately,. S-equol induced cell cycle arrest at the G0/G1 phase in Ln CaP cells(p53 wt) and DU145 cells(p53 mt) and induced cell cycle arrest in the G2/M phase in PC3 cells(p53 null), indicating that its cell cycle arrest effects may be associated with the p53 status of the prostate cancer cells. The results of Western blot analyses indicated that S-equol increased the FOXO3 a protein expression and decreased the MDM2 protein expression. In PC3 cells, S-equol exposure decreased the expression p-AKT and p-FOXO3 a level and increased expression of the FOXO3 a downstream proteins, including p21, p27, Fas L, and Bim. After S-equol treatment, the FOXO3 a protein level in the nucleus was significantly increased based on the immunofluorescence assay. We also demonstrated the induction of apoptosis and cell cycle arrest of S-equol after the cells treated with Wortmannin, an AKT inhibitor. 10 mg/mL of Wortmannin increased S-equol-induced apoptosis and G2/M phase arrest in PC3 cells; the level of FOXO3 a proteins in nucleus was significantly higher than that of S-equol treatment only. In the PC3 xenograft tumor model, at doses of 10 mg/kg and 20 mg/kg bodyweight, S-equol led to remarkable tumor growth inhibition by 43.2% and 28.4%, compared with control animals treated with the vehicle.Conclusion: Our data from the present study have demonstrated that S-equol had significant anti-prostate cancer activities in vitro and in vivo and that its anticancer effects was most likely to be associated with activating FOXO3 a via AKT specific pathway and inhibitory effect on MDM2 expression. We believe that the results from the present study not only help better understand the molecular mechanisms of this unique secondary metabolite of natural product anti-cancer compound, but also provide a basis for the development of daidzein and its analogs as novel anticancer agents. |
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