Abstract
Genistein, the major isoflavone in soybean, has been reported to exert anticancer effects on various types of cancer including ovarian cancer; however, its chemopreventive effects and mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously developing ovarian cancer models. In this study, we demonstrated the preventive effects and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer at high incidence rates. Laying hens were randomized to three groups: control (3.01 mg/hen, n = 100), low (52.48 mg/hen n = 100), and high genistein supplementation (106.26 mg/hen/day; per group). At the end of 78 weeks, hens were euthanized and ovarian tumors were collected and analyzed. We observed that genistein supplementation significantly reduced the ovarian tumor incidence (P = 0.002), as well as the number and size of the tumors (P = 0.0001). Molecular analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde, a marker for oxidative stress and the expression of NFκB and Bcl-2, whereas it upregulated Nrf2, HO-1, and Bax expression at protein level in ovarian tissues. Moreover, genistein intake decreased the activity of mTOR pathway as evidenced by reduced phosphorylation of mTOR, p70S6K1, and 4E-BP1. Taken together, our findings strongly support the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of the genistein on the molecular pathways involved in ovarian tumorigenesis.
Introduction
Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer-related mortality among women in the United States. An estimated 22,280 new cases of ovarian cancer and 14,240 deaths (accounting for 5% of cancer-related deaths among women) are expected in the United States in 2016 (1). Ovarian cancer is a molecularly and histopathologically heterogeneous disease associated with risk factors including family history of breast or ovarian cancer, age at diagnosis, race, and smoking (2–4). The current standard of care for ovarian cancer involves cytoreductive surgery followed by adjuvant chemotherapy (5). However, the lack of reliable screening tests for detection of ovarian cancer at early stages, high rate of recurrence after surgery, and resistance to available chemotherapeutic drugs lead to poor prognosis and high mortality, with an overall 5-year survival rate of 46% (1, 6–8). Therefore, there is an urgent need for novel therapeutic agents that target specific molecular defects and have the potential to prevent ovarian cancer and improve outcomes for patients with ovarian cancer.
Epidemiologic studies have demonstrated that the incidence of ovarian cancer, as those of breast and prostate cancers is much lower in Asian countries where soy foods are consumed in larger amounts compared with Western countries, suggesting the association between high dietary intake of soy isoflavones and reduced risk of ovarian cancer (9–13). As the major biologically active isoflavone in the soy diet, genistein has been extensively investigated for its chemopreventive and chemotherapeutic potential in various types of cancer. Genistein is a naturally occurring nonsteroidal plant compound with a structural similarity to the steroid hormone estradiol (17β-estradiol) that functions as selective estrogen receptor modulators (14–16). Several studies have shown that genistein inhibits ovarian carcinogenesis through pleiotropic molecular mechanisms by targeting multiple signaling pathways associated with the hormonal activity, cell cycle, apoptosis, angiogenesis, and metastasis (17–22). In addition, genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in ovarian cancer cells (23, 17).
Although a number of mouse models for human ovarian cancer have been developed, the nonspontaneous nature of these models and the dissimilarities in the histopathology of ovarian cancer between mouse and human limit the clinical relevance, leading to an inappropriate animal model to study human ovarian cancer (24). On the other hand, the laying hen, which is the only nonhuman animal that spontaneously develops ovarian cancer with a high prevalence, provides a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25). The key similarities between the ovarian cancer in the hen model and the one in human include epidemiologic, histologic, and molecular characteristics, supporting the laying hen as a relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects of novel agents on the disease (25–30). In the light of these recent findings, we investigated the effects of genistein on spontaneous ovarian cancer using the laying hen model, providing further mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian cancer.
Materials and Methods
Animals and experimental design
A total of 300 brown laying hens (104 weeks old; ATAK-S hybrid, Gallus domesticus) were used for the study in accordance with animal welfare regulations and under the Guide for the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station (Elazig, Turkey). The animal protocol was approved by the Institutional Animal Care and Use Committee at the Ankara Poultry Research Station (Elazig, Turkey). Hens were fed either a basal diet containing 16.83% crude protein or 11.15 MJ/kg of metabolizable energy and 22.39 mg of genistein/kg of diet or the basal diet reconstituted with addition of 400 mg or 800 mg of genistein per kilogram of diet at the expense of corn. The genistein contained 98% glycone and 2% starches as a carrier (Bonistein, DSM Nutritional Products). Daily total diet intake was 134.3, 133.6, and 133.2 g/day per animals in control, low, and high genistein groups, respectively. Animals received genistein 3.01, 52.48, and 106.26 mg/hen/day in control, low genistein, and high genistein groups, respectively. The dosage was chosen based on previously reported dosage in poultry (31, 32). The nutrient composition of the standard diet is listed in Supplementary Table S1. Diets were prepared in batches and stored in black plastic containers at 4°C to avoid photooxidation. The birdhouse was set to a 16L:8D cycle. Water and diets were offered for ad libitum consumption throughout the experiment. The animal experiment lasted 78 weeks (from 104 to 182 weeks).
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein and centrifuged at 3,000 × g for 10 minutes for obtaining serum. After hens were euthanized, ovaries and surrounding tissues were removed, and the morphologic and histologic changes were evaluated and compared. Tumor incidence and sizes and were measured. Tumor types were determined by histologic examination using hematoxylin and eosin (H&E) staining of tissue sections. Ovarian tumors were identified as strictly cellular masses confined to the ovary. Ovarian tumor rates were presented in this study.
Tissue and serum samples and tumor tissues were immediately frozen and stored at −80°C until analysis. Tissue samples were fixed in 10% neutral-buffered formalin, routinely processed for histology, and embedded in paraffin. Tissue blocks were used to prepare sections (6 μm) were cut. The slides were stained with H&E and were evaluated on the basis of the histopathology classification system listed in Table 1 (26, 33, 34).
Adenocarcinoma . | Characteristics . |
---|---|
Grade 1 | Well-differentiated, mitosis rare to absent, defined pattern, most common classification |
Grade 2 | Intermediate differentiation, mitosis rare to occasional, tubular pattern present but not distinct |
Grade 3 | Poorly differentiated, mitosis common, cells highly anaplastic, least common classification |
Adenocarcinoma . | Characteristics . |
---|---|
Grade 1 | Well-differentiated, mitosis rare to absent, defined pattern, most common classification |
Grade 2 | Intermediate differentiation, mitosis rare to occasional, tubular pattern present but not distinct |
Grade 3 | Poorly differentiated, mitosis common, cells highly anaplastic, least common classification |
Analysis of serum levels of genistein by high-performance liquid chromatography
At the end of the study, blood samples were collected from 12 birds randomly chosen from each treatment group. Blood samples were centrifuged at 3,000 × g for 10 minutes, and sera were collected. Sera samples were kept on the ice and protected from light until they were processed to prevent any artefactual oxidation during the experiments. Samples were stored at −80°C until analysis. Serum genistein and daidzein concentrations were measured by high-performance liquid chromatography (HPLC; Shimadzu) using Shimadzu Fluorescence RF-10 AxL Detector, and C18− ODS-3, 5μm, 4.6 × 250 mm column. The serum isoflavone (genistein and daidzein) levels were measured by the method of our previous study (35). To 200 μL of serum were added 200 μL of b-glucuronidase type H-5 solution (Sigma Chemical) in 0.2 mol/L sodium acetate buffer, pH 5.0 (3,500 units of b-glucuronidase and 193 units of sulfatase). The mixture was incubated at 37°C in a shaking water bath for 2 hours and then treated with 3,600 μL of methanol/acetic acid (95/5, vol/vol). The mixture was vortexed for 30 seconds, sonicated for 30 seconds, vortexed again for 30 seconds, and centrifuged for 15 minutes at 4°C and 800 × g. The supernatants were evaporated. We then dissolved the sample with 80% methanol at the same volume of serum. Elution was performed at a flow rate of 1 mL/minute using the following linear gradient: methanol:acetic acid (95:5, vol:vol; A), water:acetic acid (95:5, vol:vol; B), and A (by vol) at 30% for 10 minutes, from 30% to 70% in 35 minutes, and from 70% to 30% in 5 minutes (35). Chemical analyses of the diet samples were performed using procedures of Association of Official Analytical Chemists (36).
Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde, a marker for oxidative stress, (n = 12) were measured using HPLC with an LC-20AD pump, SIL-20A autosampler, SPD-20A ultraviolet-visible spectroscopy detector (at C18-ODS-3V and 5 μm with a 4.6 × 250 mm column), and CTO-10ASVP Column Oven (Shimadzu) as described previously (37). Tissue samples (300 μL) were homogenized in a mixture of 200 μL of HClO4 (0.5 mol/L) and 100 μL of 500-ppm 2[6]-di-tert-butyl-p-cresol. Next, the samples were centrifuged, and supernatants were injected (injection volume, 20 μL) into an HPLC system. The mobile phase was 30 mmol/L KH2PO4-methanol (82.5 + 17.5, v/v%, pH 3.6), the flow rate was 1.2 mL/minute, and detection at 250 nm.
Western blot analysis
Western blot analysis was performed as described previously (38). Proteins were extracted from ovarian tumor samples and were homogenized at 1:10 (w/v) in 10 mmol/L Tris-HCl buffer at pH 7.4 containing 0.1 mmol/L NaCl, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 5 μmol/L soluble soybean powder (Sigma) as a trypsin inhibitor. Samples underwent centrifugation at 15,000 × g at 4°C for 30 minutes for obtaining a supernatant. Supernatants were mixed with Laemmli sample buffer and boiled for 5 minutes. Aliquots containing 20 μg of protein were subjected to 10% SDS- PAGE and subsequently transferred to nitrocellulose membranes (Schleicher & Schuell Biosciences). Nitrocellulose membranes were washed twice for 5 minutes in PBS and blocked with 1% BSA in PBS for 1 hour prior to application of primary antibodies. Antibodies against NFκB, Bcl-2, Bax, p-mTOR, p-P70S6k, p-4E-Bp1, nuclear factor erythroid 2 (Nrf2), and heme oxygenase 1 (HO-1) were diluted at 1:1,000 in the buffer containing 0.05% Tween-20 and used. Membranes were striped and used blotted for with other antibodies. All antibodies were purchased from Abcam.
The nitrocellulose membrane was incubated at 4°C with antibodies overnight. Western blots were washed and incubated with horseradish peroxidase–conjugated goat anti-mouse IgG (Abcam). Specific binding was detected using diaminobenzidine and hydrogen peroxide as substrates. Protein loading was controlled using an anti-β-actin antibody (Sigma). Samples were analyzed in quadruplicate under each experimental condition, and protein levels were measured densitometrically using the image analysis software program ImageJ (NIH, Bethesda, MD).
Statistical analysis
Tumor incidences in the control and experimental groups were evaluated statistically using the χ2 test. Data were analyzed via ANOVA using the general linear model with the SAS program (2002; SAS Institute Inc.) to determine the effects of genistein supplementation on tumor size, protein expressions, and serum metabolites. When a significant F statistic (P ≤ 0.05) in the ANOVA was noted, the least squares mean procedure was performed to separate means that were significantly different (P < 0.05). Linear and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three dosages of genistein administered to the animals for serum metabolites.
Results
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of spontaneous ovarian tumors, a total of 300 laying hens at age of 182 weeks were randomized to three groups (n = 100 per group): (i) control (3.01 mg/day genistein), (ii) low-dose genistein (LG; 52.48 mg/hen/day), and (iii) high-dose genistein (HG; 106.26 mg/hen/day; Supplementary Fig. S1). At the end of 78 weeks administration of genistein, the study was terminated and necropsy was performed for the examination of gross pathology and microscopy of the tumors (Figs. 1 and 2). Hens revealed a significant effect of genistein on the incidence of spontaneous ovarian cancer (Table 2). Thirty percent of the hens in the control group developed ovarian tumors as reported previously (25). The incidence of ovarian cancer significantly decreased in both LG- and HG-treated hens (19% and 10%, respectively) compared with control group (P = 0.002), indicating that genistein acts in a dose-dependent manner. On the basis of the histopathologic assessment of the tumors, two subtypes of ovarian cancer including serous and mucinous carcinomas were observed in these hens; representative images are illustrated in Fig. 2B and C. The histopathology grading system based on mitotic developments and cellular differentiation is illustrated in Table 1 for the ovary. There is no significantly lower incidence of adenocarcinoma in birds receiving genistein compared with the control birds (P > 0.05). In the control group, 63% of tumor-bearing hens developed serous carcinoma, whereas 37% of them had mucinous carcinoma. However, there was no significant difference in the incidence of each subtype of ovarian tumors between control and treatment groups (Table 2).
. | Dietary genistein levels, mg/hen per day . | –P– . | ||
---|---|---|---|---|
Item . | 3.01 . | 52.48 . | 106.26 . | X2 . |
Tumor (%) | 30/100 | 19/100 | 10/100 | 0.002 |
Incidence | 30 | 19 | 10 | χ2 = 12.701 |
Reduction | — | −36.7% | −66.7% | |
Serous carcinoma (%) | 19/100 | 11/100 | 6/100 | 0.017 |
Incidence | 19% | 11% | 6% | χ2 = 8.144 |
Reduction | — | −42.1% | −68.4% | |
Mucinous carcinoma (%) | 11/100 | 8/100 | 4/100 | 0.175 |
Incidence | 11% | 8% | 4% | χ2 = 3.485 |
Reduction | — | −27.3% | −63.6% | |
Adenocarcinoma incidence (%) | ||||
Grade 1 | 5/30 | 7/19 | 5/10 | 0.084 |
16.7% | 36.8% | 50.0% | χ2 = 4.943 | |
Grade 2 | 18/30 | 8/19 | 3/10 | 0.196 |
60.0% | 42.1% | 30.0% | χ2 = 3.258 | |
Grade 3 | 7/30 | 4/19 | 2/10 | 0.968 |
23.3% | 21.1% | 20.0% | χ2 = 0.064 | |
Number of tumorsa | 37/100 | 19/100 | 10/100 | 0.0001 |
Size/hen, mma | 2.81 ± 0.50b | 1.09 ± 0.27b | 0.47 ± 0.16b | 0.0001 |
Size/hen with tumors only, mm1 | 6.86 ± 0.85 | 5.26 ± 0.81 | 4.70 ± 0.67 | 0.249 |
Size range of tumors, mm | 1–22 | 1–12 | 2–8 | — |
Survival (%) | 83/100 | 88/100 | 91/100 | 0.228 |
83 | 88 | 91 | χ2 = 2.953 |
. | Dietary genistein levels, mg/hen per day . | –P– . | ||
---|---|---|---|---|
Item . | 3.01 . | 52.48 . | 106.26 . | X2 . |
Tumor (%) | 30/100 | 19/100 | 10/100 | 0.002 |
Incidence | 30 | 19 | 10 | χ2 = 12.701 |
Reduction | — | −36.7% | −66.7% | |
Serous carcinoma (%) | 19/100 | 11/100 | 6/100 | 0.017 |
Incidence | 19% | 11% | 6% | χ2 = 8.144 |
Reduction | — | −42.1% | −68.4% | |
Mucinous carcinoma (%) | 11/100 | 8/100 | 4/100 | 0.175 |
Incidence | 11% | 8% | 4% | χ2 = 3.485 |
Reduction | — | −27.3% | −63.6% | |
Adenocarcinoma incidence (%) | ||||
Grade 1 | 5/30 | 7/19 | 5/10 | 0.084 |
16.7% | 36.8% | 50.0% | χ2 = 4.943 | |
Grade 2 | 18/30 | 8/19 | 3/10 | 0.196 |
60.0% | 42.1% | 30.0% | χ2 = 3.258 | |
Grade 3 | 7/30 | 4/19 | 2/10 | 0.968 |
23.3% | 21.1% | 20.0% | χ2 = 0.064 | |
Number of tumorsa | 37/100 | 19/100 | 10/100 | 0.0001 |
Size/hen, mma | 2.81 ± 0.50b | 1.09 ± 0.27b | 0.47 ± 0.16b | 0.0001 |
Size/hen with tumors only, mm1 | 6.86 ± 0.85 | 5.26 ± 0.81 | 4.70 ± 0.67 | 0.249 |
Size range of tumors, mm | 1–22 | 1–12 | 2–8 | — |
Survival (%) | 83/100 | 88/100 | 91/100 | 0.228 |
83 | 88 | 91 | χ2 = 2.953 |
aData are presented as the means and SEs.
bMeans in the same line without a common superscript differ significantly (P < 0.05).
In addition, genistein treatment significantly reduced both the number and size of ovarian tumors compared with the control group (Table 2). Hens in the control group had an average of 37of 100 tumors (37 tumors in total number of animals 100), whereas low- and high genistein–treated hens had 19of 100 and 10 of 100 tumors, respectively. Average sizes of the tumors were 2.81, 1.09, and 0.47 mm in the control, LG, and HG groups, respectively (P = 0.0001).
At the end of 78 weeks administration of genistein, we also observed that overall survival rates in genistein groups were higher compared with that of control group although the differences were not statistically significant (Table 2). In control group, 83% of the hens stayed alive until the end of the experiment while 88% and 91% of the animals were alive in the LG and HG groups, respectively.
Genistein supplementation results in enhanced genistein levels in serum and reduced malondialdehyde in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was specifically due to the genistein intervention, we measured serum levels of genistein and daidzein in 12 hens per group using HPLC (Fig. 3A and B). As expected, hens treated with low- or high genistein–fed groups had significantly higher serum genistein levels (251.83 nmol/L and 358.00 nmol/L, respectively) compared with the control animals (148.75 nmol/L; P < 0.05), and the increase in the level of serum genistein was dose-dependent. However, genistein supplementation had no effect on serum level of daidzein in any of the treatment groups (Fig. 3B).
Several studies have shown that oxidative stress is involved in a wide variety of cancers including ovarian cancer (35). To determine the effects of genistein on the hen ovary in the context of oxidative stress, we analyzed the levels of malondialdehyde, which is a widely used marker of oxidative stress, in the ovaries of 12 hens per group using HPLC (Fig. 3C). Our data showed that treatment of hens with genistein significantly and dose-dependently decreased the level of malondialdehyde in the ovary (P < 0.05). The average level of ovarian malondialdehyde was 3.04 nmol/mg in the control group while it was 1.99 nmol/mg and 1.21 nmol/mg in low- and high genistein–fed groups, respectively. These results confirm that genistein treatment could ameliorate oxidative stress in the hen ovary.
Genistein decreases the expression of NFκB and Bcl-2 while increasing the expression of Bax in the ovary of laying hens
To characterize the molecular mechanisms underlying the genistein-induced changes in the pathogenesis of ovarian cancer, we first examined the effect of genistein on the NFκB signaling pathway, which has been shown as one of the key survival pathways activated by oxidative stress (40). As illustrated in Fig. 4A, both LG and HG treatments significantly reduced the protein expression level of NFκB, indicating that genistein mediates its antitumor effects on ovarian cancer through NFκB signaling pathway. In addition, the expression levels of prosurvival Bcl-2 and proapoptotic Bax, which are transcriptionally regulated by NFκB, were also analyzed (Fig. 4B and C). Our findings showed that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax, leading to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis.
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the ovarian carcinogenesis, we studied the effects of genistein on the mTOR survival signaling which has also been shown to be associated with oxidative stress (41). mTOR, which is a downstream target of AKT, is a Ser/Thr kinase that phosphorylates p70S6K and 4EBP1 (42). As shown in Fig. 5A–C, treatment of hens with low or high genistein significantly decreased the levels of phosphorylated proteins of mTOR, p70S6K, and 4EBP1, leading to the inactivation of the mTOR signal transduction. These data demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in ovarian cancer cells.
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2–related factor 2 (Nrf2) is one of the major mechanisms in the cellular defense against oxidative stress (43). Similar to Nrf2, its downstream protein HO-1 has also a protective effect against oxidative stress (44). As illustrated in Fig. 6A and B, significantly increased levels of Nrf2 and HO-1 were observed in the genistein-treated groups, indicating that genistein exerts its chemopreventive effect on the ovary by activating the Nrf2-induced cellular stress responses.
Discussion
Although surgical and chemotherapeutic interventions have improved the overall survival rates, effective treatment of ovarian cancer is limited due to the major challenges, such as clinicopathologic and genetic heterogeneity, lack of early detection strategies for the disease, tumor recurrence, and resistance to conventional chemotherapeutic drugs (6–8, 45). Therefore, chemoprevention of ovarian cancer by nontoxic, naturally occurring or synthetic agents provides a rational approach to reduce the incidence and mortality rates of ovarian cancer. Epidemiologic studies have shown that the dietary intake of soybean is associated with reduced risks of various types of cancer, including ovarian cancer (46). Genistein, the most abundant isoflavone in soybean, has been reported to play a key role in the prevention of ovarian cancer (17–23). Although numerous epidemiologic and in vitro studies have demonstrated that genistein is an effective antitumor agent in the chemoprevention of ovarian cancer, there is a lack of well-characterized studies that address the efficacy and mechanisms of action of genistein in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis. In this study, therefore, we utilized the laying hen model to prospectively test the chemopreventive effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the molecular mechanisms underlying the actions of genistein on the initiation and progression of ovarian cancer.
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate, providing an appropriate natural experimental model of human ovarian cancer (25). The fact that laying hens and humans share some similarities in reproductive physiology and hens have also high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-induced DNA damage to ovarian cells in both hen and human ovarian cancers (47). Histologic classification of ovarian tumors based on the tumor stage and grade have indicated that similar to humans, four subtypes, including serous, endometrioid, mucinous, and clear cell carcinomas, are observed in hens (26). Recent studies have reported that several biomarkers of human ovarian cancer are also expressed in ovarian tumors of hens, including cytokeratin, EGFR, cytochrome P450 family member CYP1B1, proliferating cell nuclear antigen, VEGF, CA125, and HER2 (28, 30, 48, 49). Moreover, as in human ovarian tumors, molecular alterations in p53 and Ras genes have been identified in ovarian tumors of hens (29). On the basis of the findings validating that ovarian cancer in the laying hen model recapitulates the etiology and disease progression in humans, hens have been previously used in studies testing the effects of chemopreventive agents such as oral contraceptives, aspirin, and flaxseed in the prevention of ovarian cancer (50, 51).
To the best of our knowledge, this study is the first study to investigate the chemopreventive effects of genistein in the laying hen model of ovarian cancer. In this pilot study, we conducted a three-armed randomized controlled trial to assess the effects of genistein intervention on the incidence of spontaneous ovarian cancer in laying hens. Our data demonstrated that genistein significantly and dose-dependently reduced the incidence rate of ovarian cancer, consistent with previously published epidemiologic and in vitro findings. In addition, we observed an increased survival rate in genistein-treated animals although the effect was not significant. Histologic analysis of the ovarian tumors revealed that two subtypes of ovarian cancer, serous and mucinous carcinomas, were observed in these hens. However, the prevalence of different subtypes did not significantly vary between control and genistein-treated hens. Genistein intake also significantly decreased the number and size of ovarian tumors in hens, indicating the inhibitory effect of genistein on the growth of ovarian cancer cells. Analysis of genistein levels in the serum showed a dose-dependent increase in hens fed with the diet containing genistein whereas there was no change in the serum daidzein levels of these animals, confirming that the tumor inhibitory effect has been linked specifically to genistein.
It is well documented that tumor initiation and progression in the ovary has been associated with chronic inflammation which is activated by oxidative stress (52). Therefore, we first assessed the level of malondialdehyde, which is a biomarker for oxidative stress, in the ovaries of the control and genistein-fed animals. Our results showed that genistein supplementation resulted in a significant dose-dependent reduction of malondialdehyde levels in the ovary, suggesting that genistein exerts its chemopreventive effects on the ovary via oxidative stress–induced signaling pathways. On the basis of this finding, we hypothesized that genistein could ameliorate oxidative stress and inflammatory responses in the ovary through regulation of NFκB, mTOR, and Nrf2 pathways which are involved in the pathogenesis of ovarian cancer.
NFκB signaling, which is a critical molecular link between inflammation and cancer, is known to regulate key processes in several malignancies, including ovarian cancer (53–55). Activation of NFκB cascade has been shown to correlate with clinical outcome in patients with ovarian cancer and is associated with growth and progression of ovarian tumors (56–58). Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NFκB inhibitors revealed that NFκB pathway regulates genes associated with cell proliferation, adhesion, invasion, angiogenesis, and the creation of a proinflammatory microenvironment, including TNF cytokine network (54). Targeting NFκB pathway is, therefore, of interest in the suppression of inflammatory processes. Our data showed that genistein supplementation significantly reduced the expression of NFκB, as well as its downstream targets Bcl-2 and Bax at the protein level, resulting in the induction of apoptosis.
It has been reported that PI3K/AKT/mTOR signaling pathway is frequently activated in ovarian cancer (59). It is a complex signaling network transducing signals from various growth factors and cytokines (e.g., EGF, heregulin, and TGF) through receptor tyrosine kinases and G protein–coupled receptors into intracellular messages by generating phospholipids, which activate downstream effectors, including AKT and mTOR, via phosphorylation (42). Once activated, mTOR phosphorylates two key translation-regulating factors, ribosomal protein S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), resulting in increased translation of target genes involved in cell cycle, cell survival, metabolism, motility, angiogenesis, chemoresistance, and genomic instability (60). In our study, we observed that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells, suggesting that genistein may have the potential to enhance the efficacy of therapeutic agents that target PI3K/AKT/mTOR signaling cascade in ovarian cancer cells.
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to protect cells against oxidative stress (61). Under basal conditions, Nrf2 is present in the cytoplasm and kept transcriptionally inactive through binding to its inhibitor, Kelch like-ECH-associated protein 1 (Keap1), which targets Nrf2 to ubiquitination and the subsequent proteasomal degradation (62, 63). In the presence of oxidative stress, the cysteine residues of Keap1 become oxidized, resulting in disruption of the Nrf2–Keap1 complex. This dissociation allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements, resulting in the transcription of its downstream target genes (64). Interestingly, aberrant activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are associated with poor prognosis in patients with ovarian cancer (65). Recent studies have demonstrated that not only healthy cells but also various cancer cells, including ovarian tumors, can protect themselves against oxidative stress by activating the transcription factor Nrf2, the master regulator of antioxidant genes, suggesting a dual role of Nrf2 in carcinogenesis (66, 67). Therefore, these findings may suggest that activation of Nrf2 could be utilized as a cancer prevention strategy, whereas inhibition of Nrf2 could be effective in cancer treatment (68, 69). Our results showed that genistein intervention significantly and dose dependently increased the expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors, indicating the involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer.
In conclusion, our findings in laying hen model of spontaneous ovarian cancer indicate that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by modulating NFκB, mTOR, and Nrf2 signaling pathways. These results provide further support and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a biologically relevant in vivo model, providing a strong rationale for clinical studies to assess the protective effects of genistein, which may ultimately lead to better clinical outcomes and improved overall survival rates for patients diagnosed with ovarian cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: K. Sahin, B. Ozpolat, E. Yenice, I.H. Ozercan, O. Kucuk
Development of methodology: K. Sahin, B. Ozpolat, M. Tuzcu, N. Sahin, I.H. Ozercan
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Sahin, B. Ozpolat, E. Yenice, C. Orhan, M. Tuzcu, N. Sahin, I.H. Ozercan
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Sahin, B. Ozpolat, B. Bilir, C. Orhan, N. Sahin, I.H. Ozercan, O. Kucuk
Writing, review, and/or revision of the manuscript: B. Ozpolat, B. Bilir, I.H. Ozercan, N. Kabil, O. Kucuk
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Sahin, B. Ozpolat, E. Yenice
Study supervision: K Sahin, B. Ozpolat
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.