Genistein suppresses the inflammation and GSK-3 pathway in an animal model of spontaneous ovarian cancer

Background/aim Numerous studies show that cancer risk is reduced by consumption of soy-based foods containing genistein, but its effects on the glycogen synthase kinase-3 pathway (GSK-3) in ovarian cancer is unknown. Therefore, we tested the properties of genistein on inflammatory biomarkers and GSK-3 signaling pathways in the ovaries of old laying hens with ovarian cancer. Materials and methods A total of 300 laying hens were distributed into three groups as follows: group 1, animals fed a standard diet (comprising 22.39 mg of genistein/kg of diet); groups 2 and 3, animals fed a standard diet reconstituted with supplementation of 400 mg or 800 mg of genistein/kg of diet, respectively. Results Genistein modulated the inflammatory biomarkers by decreasing serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) compared with control (p < 0.001). Moreover, it upregulated insulin receptor substrate-1 (p-IRS-1) and protein kinase B (p-AKT), but downregulated GSK-3α and β after treatment. It acts in a dose-dependent manner. Conclusion Genistein exhibited an anticancer effect by reducing proinflammatory biomarkers levels and inhibiting GSK-3 expression in the ovaries of old laying hens. It is a potential candidate in the chemoprevention and/or treatment of ovarian cancer.


Introduction
Ovarian cancer has the third highest incidence of gynecological cancer in women globally, and the second most common gynecological cancer mortality [1]. The incidence and mortality of ovarian cancer show remarkable geographic variations at higher rates in industrialized countries. For instance, incidence and mortality in high/ very high human development index (HDI) districts are approximately 7.0 and 3.8 per 100,000 women and 5.7 and 4.0 for 100,000 women in low/medium HDI regions, respectively [2]. Lifestyle factors and genetic predisposition are the main risk factors for ovarian cancer development [3]. The etiology of this disease is poorly understood. The key principles are continuous ovulation and gonadotropin overproduction [4]. Malignant transformation may result from overexcitation due to hormonal factors, including estrogen-rich follicular fluid after erosion or extreme gonadotropin levels leading to stimulation via estrogen or estrogen precursors [4]. The high mortality rate of ovarian cancer is linked to difficulties in diagnosis of the early stages of the disease, high rate of recurrence, platinum resistance, and inflammation [5]. Glycogen synthase kinase-3 (GSK-3), an enzyme to regulate glycogen synthesis, has a key role in regulating immune and inflammatory responses by activities of various transcription factors that have been well reported [6].
Inflammation, oxidative stress, and DNA damage are causative factors in carcinogenesis [7]. Ovulation itself has been reported to be related to inflammation at the epithelial and follicle level [8]. In addition, talc exposure, endometriosis, and pelvic inflammatory diseases are other risk factors for developing ovarian cancer, but they do not directly affect ovulation and sex hormone profile [9]. Modern treatment of ovarian cancer is associated with drug resistance, a high risk of recurrence, and severe side effects in some cases [10]. Thus, the investigation for natural compounds with fewer side effects and easy availability is on demand. Studies have shown that high isoflavones content in soy products prevents ovarian cancer [11]. In addition, high soy oil consumption significantly decreased the occurrence of ovarian cancer in women [12]. Isoflavones, a major class of phytoestrogens, are plant-derived compounds present in soy products and other legumes. Genistein (5,7,4'-trihydroxyisoflavone), the most commonly studied natural isoflavone, has been shown to affect the cell cycle of cancer cells progression and apoptosis [13]. In SK-OV-3 cells, genistein prevents cellular proliferation and induces cell cycle hold at the G2/M phase in a dose-and time-dependent manner [14]. In addition, it has been shown that genistein downregulates vascular endothelial growth factor (VEGF) receptors, which are considered critical targets for ovarian cancer treatment. Genistein is compared to other isoflavones, with a potent inhibitory property on VEGF protein secretion [15]. In ovarian cancer cells, high concentrations of genistein induce apoptosis and cell death while low concentrations exhibit antioxidant potential without any cytotoxic effect [16].
To evaluate the effects of genistein in ovarian cancer, laying hens are suitable experimental models because they spontaneously stimulate ovarian cancer largely when stopping egg production similar to women. However, other animals such as mammals and rodents do not spontaneously develop ovarian cancer [17]. In addition, the risk of developing ovarian cancer in chickens as well as in women increases with age and the number of ovulation for life [18]. The high prevalence of ovarian cancer in laying hens depends on age, genetic predisposition, reproductive history, and diet. Due to the similarity between ovarian cancer in laying hens and human ovarian cancer, laying hen is a proper experimental animal model for translational research [19]. This study examined the supplemental genistein properties on inflammatory biomarkers and glycogen synthase kinase-3 signaling pathway in laying hens with ovarian cancer.

Animals and design
Three-hundred laying hens (104-week-old; ATAK-S hybrid, Gallus domesticus) were used in this work. The Animal Experimental Ethical Committee of Poultry Research Station (Ankara, Turkey) permitted the study. The birds were divided into three groups (n = 100) and treated as follows: group 1, animals fed a standard diet containing 16.83% crude protein (CP), 11.15 MJ/kg metabolizable energy (ME), and 22.39 mg genistein per kg of diet (Table); groups 2 and 3, birds fed a standard diet reconstituted with the adding of 400 mg or 800 mg of genistein per kg of standard diet at the expense of corn, respectively. The genistein (Bonistein™, 98% aglycone, and 2% starches as a carrier) was provided by DSM Nutritional Products Inc. (İstanbul, Turkey). Animals received 3.01, 52.48, and 106.26 mg genistein/hen per day in the control, low genistein, and high genistein groups, respectively. The birds had food and water ad libitum during the experimental period (78 weeks).

Molecular analyses
For this purpose, Western blot analyses were done as defined previously [17].

Statistical analysis
All data were shown as the mean ± standard error of the mean. Data were evaluated by analysis of variance using the General Linear Model with the SAS program (SAS Institute Inc.). When a significant F statistic (p < 0.05) was noted, the least-squares mean procedure was performed to differentiate between tools that were significantly different (p < 0.05).

Effects of genistein on protein expression levels in ovarian samples
Both moderate (52.48 mg) and high (106.26 mg) genistein supplementation significantly reduced the protein expression levels of IL-6, IL-8, VEGF, and TNF-α in the ovarian samples, indicating that genistein mediates its antiinflammation effects on ovarian cancer through an inflammation signaling pathway. Genistein acts in a dosedependent manner (Figures 2A-2D).
As illustrated in Figures 3Aand 3B, a significant increase in insulin receptor substrate-1 (p-IRS-1) and p-AKT levels were observed in the genistein-treated groups, with a remarkable effect recorded in animals administered with the highest dose (106.26 mg). On the contrary, genistein supplementation induced a significant reduction in the protein of GSK-3α and β levels compared with the control group ( Figures 3C and 3D).

Discussion
Different experimental and clinical studies propose a preventive role of genistein on several cancers such as colon [20], liver [21], thyroid [22], prostate [23], breast [24], and ovarian cancers [10]. In some patients, advanced therapy of ovarian cancer is related to drug resistance, increased risk of disease recurrence, poor outcome, and severe side effects [10]. Therefore, further research is needed for natural compounds with fewer side effects and easy availability. Frequent consumption of soy products has been recognized to decrease the risks of developing ovarian cancer, mainly due to the high content in an isoflavone called genistein [10]. In the current study, we assessed the properties of genistein on inflammatory biomarkers and glycogen synthase kinase-3 signaling pathway in the ovaries of old laying hens with ovarian cancer. Laying hens, which have been shown to have similar features with humans, develop spontaneous ovarian cancer at a high rate, supplying a favorable experimental model for human ovarian cancer [17]. Indeed, laying hens have shown that ovarian cancer pathogenesis is related to ovulation-induced DNA damage in ovarian cells, similar to humans [25]. Ovarian tumors of laying hens have been classified into four subtypes in terms of tumor stage and grade, including mucinous, clear cell, and serous carcinomas, as those of humans [26]. Recently, the same experimental model has been used by our research team [17].
In the present study, genistein supplementation significantly lowered TNF-α, IL-6, and IL-8 levels in serum compared with the control animals. It has been demonstrated that tumor initiation and progression in the ovary is linked to chronic inflammation activated by oxidative stress [27]. Indeed, tumor cell proliferation and migration stimulate inflammatory cytokine production, including IL-6, IL-8, and TNF-α [28]. The reduction of inflammation biomarkers observed in the current study indicates an antiinflammatory property of genistein in the ovary of old laying hens. This result is similar to that reported by Li and Zhang [29] and Spagnuolo et al. [30], who demonstrated that genistein could suppress inflammatory mediators such as IL-6, IL-8, and TNF-α. Because of the importance of inflammation on cancer occurrence and progression, genistein could be a potential therapeutic agent for ovarian cancer treatment.
The vascular endothelial growth factor (VEGF) is a member of proteins, performing many endothelial cell functions, such as lymphangiogenesis [31]. VEGF-C is the main factor supporting the metastasis of cancer by activating the VEGF-C/VEGF-R3 signaling pathway and increasing cell mobility and invasiveness [18]. Because VEGF-C signaling affects tumor cells directly, the treatment of ovarian cancer is also mediated by decreasing VEGF-C levels. In the present study, genistein supplementation induced a significant decrease in VEGF-C expression level after treatment. This result corroborates with the previous finding reported by Hu et al. [32], who demonstrated that genistein reduced the expressions of VEGF-C levels in the synovial tissue. The importance of VEGF-C level reduction and the mediation of the treatment via the VEGF-C statements would be controversial, considering the main effects of bevacizumab on the VEGF-A levels rather than VEGF-C levels. Both the paclitaxel and bevacizumab could induce VEGF-C expression, which could be related to tumoral escape [33,34]. Moreover, the anticancer property of genistein observed in the current study is similar to that reported previously by our research group with curcumin in a spontaneously developing hen ovarian cancer model [17].
A possible molecular target for cancer treatment is GSK-3, an extremely conserved serine/threonine kinase [35]. The hyperactivation of GSK-3 may function as an oncogene in different types of cancers, including colon cancer [36], and ovarian cancer [37]. On the contrary, previous reports indicate that GSK-3 inhibitors are capable to suppress the proliferation of malignant cells [38]. Since protein kinase B (PKB or AKT) is a key regulator of protein translation, transcription, cell proliferation, and apoptosis, it is also considered a possible target for cancer prevention/ treatment [39]. The activity of GSK-3β is decreased by the phosphorylation of Ser9 and several studies have shown that Ser9 in GSK-3β is phosphorylated and inactivated by AKT [40]. IRS signals proteins that act as intermediates of stimulated cell surface receptors, most remarkably for the insulin and insulin-like growth factor receptors [41]. It has been reported that IRS proteins interface with numerous signaling pathways usually associated with tumor progress  Blots were repeated at least 3 times (n = 3) and a representative blot is shown. β-actin was included to ensure equal protein loading. *p < 0.05, **p < 0.01 statistically different compared with the control group. TNF-α: tumor necrosis factor-alpha; IL-6: interleukin 6; IL-8: interleukin 8; VEGF-C: vascular endothelial growth factor-C. Blots were repeated at least 3 times (n = 3) and a representative blot is shown. β-actin was included to ensure equal protein loading. *p < 0.05, **p < 0.01, ***p < 0.001 statistically different compared with the control group. IRS-1: insulin receptor substrate 1; AKT: protein kinase B; GSK-3α and β: glycogen synthase kinase-3 alpha and beta. and progression by impacting cell metabolism, motility, survival, and proliferation [41]. In cultured bovine adrenal chromaffin cells, Sugano et al. [42] presented that the rise of Ser9-phosphorylation of GSK-3β was followed by reducing IRS-1 and IRS-2 levels. The significant increase in p-IRS-1 and p-AKT expression levels after treatment with genistein may justify the low level of GSK-3 expression. Because the expression of GSK-3 is significantly higher in ovarian carcinoma tissues [43], the significant reduction in GSK-3α and β expression in the current study after treatment with genistein indicates an anticancer effect.
In conclusion, the data presented demonstrate that genistein supplementation exhibited an anticancer effect by reducing inflammatory biomarkers and modulating GSK-3 signaling pathway in the ovaries of laying hens. Genistein is a potential candidate in the chemoprevention and/or treatment of ovarian cancer.