Pro-apoptotic and anti-angiogenic actions of 2-methoxyestradiol and docosahexaenoic acid, the biologically derived active compounds from flaxseed diet, in preventing ovarian cancer

Ovarian cancer is one of the deadliest gynecological cancers, ranking fifth in all cancer-related deaths in women. Unavailability of suitable predictive biomarkers make the disease hard to detect until stage III/IV leading to its poor prognosis and fewer treatment options. Consequently, the 5-year survival rate for all epithelial types of ovarian cancer in the United States is currently 47%; the estimated number of ovarian cancer cases to be diagnosed in 2018 is 22,240 and the number of estimated deaths is 14,070 in United States, per SEER cancer statistics [1].

The laying hen provides the only animal model that develops the disease naturally. Histologically, the disease closely resembles the human form of the disease [2‐4]. Expression of different molecular markers [5‐8], symptoms such as profuse ascitic fluid and peritoneal metastasis in stage III/IV of the disease [7, 9], are found to be very similar to the human disease. Ovarian cancer is also directly correlated to the number of ovulations during the lifespan in both women and hens. A woman potentially ovulates around 400 times prior to menopause, and the average age of diagnosis is 63. Chickens start laying eggs approximately by the age of 5 months, and thus ovulate around 400 times by their 2nd year of lay [2]. By two and a half years, a significant number of hens will have developed ovarian cancer. Reducing the number of ovulations in hens [10, 11] has been shown to decrease the incidence of ovarian cancer, a phenomenon also observed in women.

Our research using the chicken model has shown that a flaxseed diet can reduce both the severity and incidence of ovarian cancer [9, 12]. Flaxseed is a rich source of omega-3 fatty acids (OM3FA), predominantly α-Linoleic acid (ALA), the phytoestrogen lignan secoisolaricisresinol diglucoside (SDG), and several other macronutrients, fiber and minerals [13]. The current study was designed to investigate the effects of the OM3FA and phytoestrogens separately. ALA is converted to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and SDG is converted to enterodiol (ED) and enterolactone (EL). DHA has been shown to have anti-inflammatory and cardioprotective properties while ED and EL are known to have anti-estrogenic and antioxidant properties [14, 15]. EL serum levels have been monitored in post-menopausal women diagnosed with breast cancer and who have received flaxseed supplements. The increase in EL in serum and disease-free survival has been positively correlated [16].

Estradiol is metabolized in the liver through its hydroxylation by three different cytochrome P450 (CYP) enzymes. CYP1A1 produces the 2-hydroxy metabolites, while CYP1B1 and CYP3A4 yield 4-hydroxy and 16-hydroxy metabolites, respectively [17]. 2-hydroxy estrogens can be readily converted into 2-methoxy [18] estrogens by catechol-O-methyltransferase enzyme (COMT) [19]. 2-methoxyestradiol (2MeOE₂) is easily excreted and known to be the least potent estrogenic metabolite while the 4-hydroxyestradiol is readily oxidized to a genotoxic compound, 3,4 quinone. Therefore, the whole flax diet favors the CYP1A1 pathway for generation of 2MeOE₂, resulting in a higher 2-hydroxy:16-hydroxy estradiol ratio, which has been shown to be protective against postmenopausal breast cancer [20].

From our previous studies, we have shown that the flax diet promotes the CYP1A1 pathway of estrogen metabolism while decreasing both CYP1B1 and CYP3A4 in the pre-neoplastic chicken ovaries. The upregulation in CYP1A1 enzyme also parallels the increase in 2-hydroxy: 16-hydroxy estradiol ratio and the 2MeOE₂ level in the serum of chickens [21]. 2MeOE₂ is an established anti-proliferative and anti-apoptotic agent [22, 23] and has been tested on different cancer cell lines over the past few years [24‐30]. Previously, we have shown that a whole flax diet promotes apoptosis in the chicken ovaries and also activation of the p38-MAPK pathway [31].

The objective of this study was to explore mechanisms of the anti-cancer effects of the flaxseed diet in ovarian carcinogenesis and explore how the biologically active components of flaxseed diets, 2MeOE₂ and DHA, accomplish their actions, focusing on two hallmarks of cancer, apoptosis and angiogenesis.

The purpose of this study was to investigate the mechanisms through which the biologically active components of flaxseed drive apoptosis and inhibit tumor angiogenesis. The present study demonstrated that 2MeOE₂ promotes apoptosis and inhibits angiogenesis in vitro and both the actions are dependent on the p38-MAPK pathway. DHA has an anti-angiogenic effect but does not induce apoptosis in ovarian cancer cells, and its effects are not p38-MAPK dependent. The effects of 2MeOE₂ and DHA were independent of ER-α expression in human ovarian cancer cells.

Previously we have shown that a whole flaxseed diet induces apoptosis in chicken ovarian tumors, promotes phosphorylation of p38-MAPK [31] and alters both estrogen signaling and metabolism in chickens. Flax diets induce CYP1A1 expression resulting in an increase in 2MeOE₂ levels in serum [31]. The OM3FA (predominantly ALA, which gets systemically converted to DHA) components of the flaxseed diet have been previously described as having potent anti-inflammatory effects in cancerous chicken ovaries by reducing prostaglandin E₂ (PGE₂) levels as well as PTGS-2 (COX-2) gene expression [12]. Taken together, these observations suggested that different components of flaxseed play different roles in the anti-cancer mechanisms of flaxseed and work in a synergistic way in the whole flaxseed diet. This study was designed to individually investigate the mechanisms of action of 2MeOE₂ and DHA, which are two principal biologically-derived compounds from the flaxseed diet, in inducing apoptosis and inhibiting angiogenesis in ovarian cancer.

Previously we have shown that a 15% flaxseed diet induces apoptosis in chicken tumors but not in normal chicken ovaries [31]. Here we investigated the effects of the lignan vs oil fractions of flax seed. The DFM diet induced apoptosis in chicken tumors but not in normal chicken ovaries. The flax oil diet did not cause a significant increase in apoptosis in normal or cancerous chicken ovaries. However, whole flax diet had a much greater pro-apoptotic effect than the DFM diet on chicken ovarian tumors, suggesting an anti-cancer entourage effect provided by the whole flaxseed-supplemented diet where the pro-apoptotic effect of the whole seed exceeds the sum of the effects of the individual components.

Immunostaining for various angiogenic markers suggested that the whole flaxseed diet exerts an anti-angiogenic effect in chicken ovarian tumors. The number of blood vessels per field of view significantly increased in ovarian tumors compared to normal ovaries in control fed hens. All three flaxseed diets have caused a significant decrease in the number of vessels in ovarian tumors with no significant changes in the normal ovaries. CD31 immunostaining across different groups showed that whole flaxseed diet significantly reduced the endothelial cell population in ovarian tumors, but had no effect on angiogenesis in normal ovarian tissues in hens. α-SMA immunostaining indicated the α-SMA positive cells decreased in ovarian tumors, as previously reported in one of our studies [32]. α-SMA is expressed in the pericytes, cells that divide and increase in number to surround and pack the newly formed vessels for vascular stabilization. Additionally, α-SMA is also expressed in cancer-associated fibroblasts (CAFs) and other smooth muscle cells The decrease of α-SMA in ovarian tumors indicates a reduction in the fibrotic condition in the ovaries of flaxseed-fed hens. One of the classic characteristics of ovarian cancer is smooth muscle metaplasia and excessive proliferation in ovarian fibroblasts resulting in a fibrotic condition [33]. This phenomenon, known as desmoplasia, contributes to the protection of the tumor cells and facilitates tumor growth by physically shielding them from the immune cells [34]. The fibrosity of the intra-tumoral stroma and the adjacent normal tissues also induces a high inflammatory burden, and diminishes the response to anti-cancer treatments. The whole flaxseed supplemented diet mediates the reduction in the fibrosity of the ovarian tumor suggesting that dietary supplementation with flaxseed could enhance the therapeutic efficacy of the treatment regimen.

However, the non-specific expression of α-SMA limited our ability to assess the maturity of the tumor vasculature. It is important to remember that tumor angiogenesis is distinctly different from growth and maturation of normal blood vessels. The hypoxic tumor micro-environment drives the growth and proliferation of the endothelial cells but the growth also renders an increase in the ratio of endothelial cells to pericytes. This results in immature, dysfunctional and leaky vessel formation. In order to evaluate the maturity of tumor angiogenesis, we immunostained the normal and cancerous ovaries with NG-2, a specific pericyte marker [35] Quantification of total number of CD31 and NG-2 positive cells per field of view indicated a decrease in pericyte to endothelial cell ratio in tumors in the control diet. The flax diets increased the ratio, indicating an improvement of the vasculature and decrease in leakiness (p > 0.05). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors expressed as a response to tissue hypoxia, and known to most effectively bind to its receptor (VEGFR-2/Flk-1) in endothelial cells promoting endothelial cell proliferation. Both VEGF and VEGFR-2 expressions significantly increased in cancer from control diet-fed chickens and decreased in cancer from chickens fed the whole flaxseed diet, with no change in normal ovarian tissues.

One of our recent works has suggested that a recombinant thrombospondin-1 type 1 repeats (3TSR) pre-treatment normalizes advanced ovarian tumor vasculature and improves the uptake of chemotherapeutic drugs [36]. Minimizing the toxic treatments as well as increasing chemotherapeutic efficacy is a desired goal in clinical oncology. Our findings in this study demonstrate that a flaxseed supplemented diet causes a reduction in angiogenesis in chicken ovarian tumors, with no significant changes in normal ovaries, thereby normalizing the vasculature of the ovarian tumors. Thus, flaxseed supplementation could be a promising combinational approach for the treatment of advanced stage ovarian carcinoma in addition to its use as a preventive therapy.

The role of ERα in the prognosis of ovarian carcinoma is controversial. Some studies have shown a correlation between ERα expression and improved outcomes in epithelial ovarian carcinoma patients [37, 38], whereas other studies show increased ERα expression decreases the overall survival of ovarian cancer patients [39, 40]. Several studies suggest that the onset of estrogen-mediated cell proliferation and cell survival leading to ovarian carcinogenesis is mediated through increased ERα expression [41, 42]. However, ERα expression also varies between different types of ovarian cancer [43]. In our previous studies, we have shown that a 15% whole flax diet significantly reduces the ERα expression in pre-neoplastic chicken ovaries [31]. Expression of ESR-2 in the chicken ovaries is very low [44] and ovarian cancer has been reported to have a higher ESR1/ESR2 ratio in both chicken and women [45‐47]. We have tested the anti-cancer actions of 2MeOE₂ on both ERα-positive (BG1) and ERα-negative (HeyC2 and TOV112D) cells and found that 2MeOE₂ exerted similar effects, regardless of ER-status. These results suggest that the anti-cancer actions of 2MeOE₂ are ER-independent.

MAPK14, or p38α, was the first identified member of the MAPK family. p38-MAPK is phosphorylated on threonine and tyrosine residues in response to stress [48]. Activation of p38-MAPK has been linked to the phosphorylation of the pro-apoptotic protein BAX, facilitating its translocation to the Mitochondrial Outer Membrane (MOM) and inducing apoptosis [49]. Phosphorylated p38-MAPK also activates a group of transcription factors such as CHOP, ATF1, p53, MEF2C and MEF2A [50, 51] that are potential mediators in the apoptotic pathway. In the current study, cells treated with 2MeOE₂ showed significant changes in viability and morphology which was partially reversed by the p38 MAPK inhibitor (SB203580) [52]. Western blots showed that the 2MeOE₂ treatment induces activation of p38-MAPK in all three cell lines and cleavage of caspase-3 paralleled phosphorylation of p38-MAPK. The cleavage of caspase-3 was significantly reduced after the cells were treated with 2MeOE₂ and SB203580, suggesting the p38-MAPK pathway contributes to the pro-apoptotic activity of 2MeOE₂. The 2MeOE₂ mediated reduction in viability of the human ovarian cancer cells was also reversed by SB203580. The in vivo angiogenesis assay showed that 2MeOE₂ exhibits an anti-angiogenic effect in a dose-dependent manner on CAM. These results agree with our previous observations that a whole flax diet increases the phospho-p38 expression in chicken ovaries and promotes apoptosis in chicken ovarian tumors [31].

Flaxseed is also one of the richest sources of OM3FA, mostly ALA. ALA is converted into longer-chain EPA and DHA. The conversion efficiency of this pathway in humans varies in terms of ethnicity and dietary habits. However, several groups have shown that a flaxseed diet increases this conversion efficiency in mammals [53]. EPA has been suggested to act as a competitive inhibitor of arachidonic acid that binds to COX-2 [54] and previously we have reported that dietary OM3FA reduces PGE₂ level in the serum and COX-2 gene (PTGS-2) expression in laying hens [12]. The effect of DHA alone on apoptosis and angiogenesis was tested in the human ovarian cancer cells. DHA has been reported to bind the peroxisome proliferator-activated receptor (PPAR) α and γ, as well as IκB, which sequesters NFκB in the cytosol, thus preventing transcription of a myriad of pro-inflammatory NFκB response genes [55]. In the current study, DHA was found not to have any pro-apoptotic effect on human ovarian cancer cells. Following a 24-h incubation, DHA treatment did not induce any caspase-3 cleavage, nor did it activate p38-MAPK which indicates that DHA effects are not mediated by p38-MAPK.

We have investigated the individual effects of 2MeOE₂ and DHA on angiogenesis by CAM assay. The chorioallantois in the chick embryo is formed in the first 4 to 5 days of embryonic development. This is when the outer mesodermal layers of the allantois and the chorion fuse together forming a connection of blood vessels that grows rapidly in the next few days. Judah Folkman first described that this window of time can be utilized for the study of angiogenesis [56]. Since then, CAM has been extensively used as an assay to study the angiogenic effects of various test compounds with different treatment methods such as filter disks, gelatin sponge, collagen implants or other gelated materials. In the present study, we have performed a filter disk based CAM assay. Both 2MeOE₂ and DHA reduced the number of sprouting vessels around the filter paper, demonstrating an anti-angiogenic effect. The effect of 2MeOE₂ is reduced by treatment with SB203580, suggesting p38-MAPK pathway plays a role in promoting the anti-angiogenic effects of 2MeOE₂. At the same time, SB203580 had no effect on the anti-angiogenic action of DHA suggesting that DHA exerts its anti-angiogenic effects in a p38-MAPK independent way.

Although the canonical signaling of p38-MAPK pathway is believed to promote proliferation and invasiveness in different cancers across different studies, there have been recent studies that have shown MAP kinases to be involved in promoting apoptosis in tumor cells. 2MeOE₂ is known to bind at or near the colchicine-binding site of the tubulin, suppressing microtubule dynamics, and thus eliciting mitotic arrest [57] in tumors both in vitro and in vivo. Recent studies with 2MeOE₂ treatment have shown that it induces JNK, Erk-1/2 and p38 activation in breast cancer cells [58], stabilizes SMAD7 and induces p38-MAPK mediated apoptosis in prostate cancer [59] and retinoblastoma cells [49]. In our previous work, we have shown that the flax diet activates p38-MAPK in chicken ovarian tumors and promotes apoptosis [31]. The current study suggests that the pro-apoptotic and anti-angiogenic effects of 2MeOE₂ are dependent on p38-MAPK, however the anti-angiogenic effect of DHA is not dependent on p38-MAPK pathway.

2-MeOE₂ has been clinically tested as an administered drug in ovarian cancer [60] and multiple myeloma patients [61], yielding promising results. We have shown that consumption of whole flaxseed in the diet promotes endogenous production of 2MeOE₂, a pro-apoptotic and anti-angiogenic compound that reduces the ovarian tumor burden, and can provide an alternative natural therapeutic modality. Flaxseed diets also provide the essential, anti-inflammatory polyunsaturated fat, DHA. Consumption of flaxseed is being tested as maintenance therapy to prevent recurrence of ovarian cancer in women {https://​clinicaltrials.​gov/​ct2/​show/​NCT02324439}. Adding flaxseed to the daily diet may be an important preventative measure in healthy women.

A flaxseed-supplemented diet, which increases the systemic production of 2MeOE₂ and DHA, induces apoptosis and decreases angiogenesis in ovarian tumors but not in normal ovarian tissues. 2MeOE₂ and DHA both have anti-angiogenic effects. 2MeOE₂ has pro-apoptotic effects but not DHA. Anti-cancer actions of 2MeOE₂ are dependent on p38-MAPK pathway, however the actions of DHA do not involve p38-MAPK. Dietary supplementation with flaxseed may help prevent ovarian cancer in women or help them live with ovarian cancer instead of die from it.