Glyceollins Trigger Anti-Proliferative Effects in Hormone-Dependent Aromatase-Inhibitor-Resistant Breast Cancer Cells through the Induction of Apoptosis.

Aromatase inhibitors (AIs) are standard treatment for estrogen-dependent postmenopausal breast tumors; however, resistance develops leading to tumor relapse and metastasis. We previously demonstrated that glyceollin inhibits proliferation, survival, and migration of hormone-independent letrozole-resistant breast cancer. Since many AI-resistant tumors remain hormone-dependent, identifying distinctions between estrogen-receptor-positive (ER+) and ER-negative (ER-) AI-resistant tumor response to therapy is critical. We hypothesize that treating ER+ letrozole-resistant T47D breast cancer cells (T47DaromLR) with a combination of 10 µM glyceollin and 0.5 µM lapatinib (a dual EGFR/HER2 inhibitor) will decrease cell proliferation through induction of apoptosis. The T47DaromLR cells were found to overexpress HER2 and MAPK while maintaining aromatase and ER levels compared to their letrozole-sensitive (T47Darom) counterparts. In the absence of estrogen stimulation, glyceollin ± lapatinib had no effect on the proliferation of the T47Darom cells, while glyceollin treatment caused 46% reduction in the proliferation of T47DaromLR cells, which was further diminished when combined with lapatinib. While neither agent influenced cell migration, glyceollin and lapatinib reduced S and G2/M phase cell entry and exclusively induced apoptosis by 1.29-fold in the T47DaromLR cells. Taken together, these results suggest that glyceollins and lapatinib may have potential as a novel combination therapeutic approach for hormone-dependent, letrozole-resistant tumors.

Characterization of Glyceollins as Novel Aryl Hydrocarbon Receptor Ligands and Their Role in Cell Migration.

Recent studies strongly support the use of the aryl hydrocarbon receptor (AhR) as a therapeutic target in breast cancer. Glyceollins, a group of soybean phytoalexins, are known to exert therapeutic effects in chronic human diseases and also in cancer. To investigate the interaction between glyceollin I (GI), glyceollin II (GII) and AhR, a computational docking analysis, luciferase assays, immunofluorescence and transcriptome analyses were performed with different cancer cell lines. The docking experiments predicted that GI and GII can enter into the AhR binding pocket, but their interactions with the amino acids of the binding site differ, in part, from those interacting with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Both GI and GII were able to weakly and partially activate AhR, with GII being more potent. The results from the transcriptome assays showed that approximately 10% of the genes regulated by TCDD were also modified by both GI and GII, which could have either antagonistic or synergistic effects upon TCDD activation. In addition, we report here, on the basis of phenotype, that GI and GII inhibit the migration of triple-negative (ER-, PgR-, HER2NEU-) MDA-MB-231 breast cancer cells, and that they inhibit the expression of genes which code for important regulators of cell migration and invasion in cancer tissues. In conclusion, GI and GII are AhR ligands that should be further investigated to determine their usefulness in cancer treatments.

The NAC family transcription factor GmNAC42-1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean.

BACKGROUND: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. RESULTS: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42-1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42-1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42-1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42-1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42-1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. CONCLUSIONS: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42-1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42-1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.

Soybean-Derived Phytoalexins Improve Cognitive Function through Activation of Nrf2/HO-1 Signaling Pathway.

As soy-derived glyceollins are known to induce antioxidant enzymes in various types of cells and tissues, we hypothesized that the compounds could protect neurons from damage due to reactive oxygen species (ROS). In order to examine the neuroprotective effect of glyceollins, primary cortical neurons collected from mice and mouse hippocampal HT22 cells were challenged with glutamate. Glyceollins attenuated glutamate-induced cytotoxicity in primary cortical neuron isolated from mice carrying wild-type nuclear factor (erythroid-derived 2)-like 2 (Nrf2), but the compounds were ineffective in those isolated from Nrf2 knockout mice, suggesting the involvement of the Nrf2 signaling pathway in glyceollin-mediated neuroprotection. Furthermore, the inhibition of heme oxygenase-1 (HO-1), a major downstream enzyme of Nrf2, abolished the suppressive effect of glyceollins against glutamate-induced ROS production and cytotoxicity, confirming that activation of HO-1 by glyceollins is responsible for the neuroprotection. To examine whether glyceollins also improve cognitive ability, mice pretreated with glyceollins were challenged with scopolamine and subjected to behavioral tests. Glyceollins attenuated scopolamine-induced cognitive impairment of mice, but failed to enhance memory in Nrf2 knockout mice, suggesting that the memory-enhancing effect is also mediated by the Nrf2 signaling pathway. Overall, glyceollins showed neuroprotection against glutamate-induced damage, and attenuated scopolamine-induced memory deficits in an Nrf2-dependent manner.

Glyceollins trigger anti-proliferative effects through estradiol-dependent and independent pathways in breast cancer cells.

BACKGROUND: Estrogen receptors (ER) α and ß are found in both women and men in many tissues, where they have different functions, including having roles in cell proliferation and differentiation of the reproductive tract. In addition to estradiol (E2), a natural hormone, numerous compounds are able to bind ERs and modulate their activities. Among these compounds, phytoestrogens such as isoflavones, which are found in plants, are promising therapeutics for several pathologies. Glyceollins are second metabolites of isoflavones that are mainly produced in soybean in response to an elicitor. They have potentially therapeutic actions in breast cancer by reducing the proliferation of cancer cells. However, the molecular mechanisms driving these effects remain elusive. METHODS: First, to determine the proliferative or anti-proliferative effects of glyceollins, in vivo and in vitro approaches were used. The length of epithelial duct in mammary gland as well as uterotrophy after treatment by E2 and glyceollins and their effect on proliferation of different breast cell line were assessed. Secondly, the ability of glyceollin to activate ER was assessed by luciferase assay. Finally, to unravel molecular mechanisms involved by glyceollins, transcriptomic analysis was performed on MCF-7 breast cancer cells. RESULTS: In this study, we show that synthetic versions of glyceollin I and II exert anti-proliferative effects in vivo in mouse mammary glands and in vitro in different ER-positive and ER-negative breast cell lines. Using transcriptomic analysis, we produce for the first time an integrated view of gene regulation in response to glyceollins and reveal that these phytochemicals act through at least two major pathways. One pathway involving FOXM1 and ERα is directly linked to proliferation. The other involves the HIF family and reveals that stress is a potential factor in the anti-proliferative effects of glyceollins due to its role in increasing the expression of REDD1, an mTORC1 inhibitor. CONCLUSION: Overall, our study clearly shows that glyceollins exert anti-proliferative effects by reducing the expression of genes encoding cell cycle and mitosis-associated factors and biomarkers overexpressed in cancers and by increasing the expression of growth arrest-related genes. These results reinforce the therapeutic potential of glyceollins for breast cancer.

Glyceollins from soybean: Their pharmacological effects and biosynthetic pathways.

Flavonoids are a highly abundant class of secondary metabolites present in plants. Isoflavonoids, in particular, are primarily synthesized in leguminous plants within the subfamily Papilionoideae. Numerous reports have established the favorable role of isoflavonoids in preventing a range of human diseases. Among the isoflavonoid components, glyceollins are synthesized specifically in soybean plants and have displayed promising effects in mitigating the occurrence and progression of breast and ovarian cancers as well as other diseases. Consequently, glyceollins have become a sought-after natural component for promoting women's health. In recent years, extensive research has focused on investigating the molecular mechanism underlying the preventative properties of glyceollins against various diseases. Substantial progress has also been made toward elucidating the biosynthetic pathway of glyceollins and exploring potential regulatory factors. Herein, we provide a review of the research conducted on glyceollins since their discovery five decades ago (1972-2023). We summarize their pharmacological effects, biosynthetic pathways, and advancements in chemical synthesis to enhance our understanding of the molecular mechanisms of their function and the genes involved in their biosynthetic pathway. Such knowledge may facilitate improved glyceollin synthesis and the creation of health products based on glyceollins.

Intestinal Absorption of Prenylated Isoflavones, Glyceollins, in Sprague-Dawley Rats.

Although prenylated isoflavones or glyceollins elicit physiological effects more potent than those by isoflavones, the bioavailability remains unclear. The present study aimed to clarify the intestinal absorption behavior of glyceollins in Sprague-Dawley rats. Upon oral administration of 1.0 mg/kg glyceollin I or III (daidzein as comparative compound) to the rats, no peaks corresponding to the intact forms of the compounds were detected in plasma by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS) analysis. In contrast, enzymatic deconjugation of plasma resulted in successful MS detection of each glyceollin; glyceollin I absorption was >10 times higher than that of daidzein, given its high log P value. The present study demonstrated for the first time that glyceollins were more absorbable than mother isoflavones due to their high hydrophobicity, and they metabolized to form sulfated, glucuronized, and methylated conjugates during the intestinal absorption process.

Enhanced biosynthesis of the natural antimicrobial glyceollins in soybean seedlings by priming and elicitation.

Glyceollins are a class of antimicrobial prenylated pterocarpans produced in soybean seedlings upon fungus elicitation. Priming with reactive oxygen species (ROS) prior to elicitation with Rhizopus oligosporus/oryzae (R) was investigated for its potential to enhance glyceollin production. ROS-priming prior to R-elicitation (ROS + R) increased glyceollin production (8.6 ± 0.9 µmol/g dry weight (DW)) more than 4-fold compared to elicitation without priming (1.9 ± 0.4 µmol/g DW). Furthermore, ROS-priming was superior to two physical primers which were used as benchmark primers, namely slicing (5.0 ± 0.6 µmol glyceollins/g DW) and sonication (4.8 ± 1.0 µmol glyceollins/g DW). Subsequently, the robustness of ROS + R was assessed by applying it to another soybean cultivar, where it also resulted in a significantly higher glyceollin content than R-elicitation without priming. ROS-priming prior to elicitation provides opportunities for improving the yield in large-scale production of natural antimicrobials due to the ease of application and the robustness of the effect across cultivars.

Induction of promising antibacterial prenylated isoflavonoids from different subclasses by sequential elicitation of soybean.

Elicited soybean (Glycine max (L.) Merrill, Leguminosae) seedlings can produce prenylated isoflavonoids from different subclasses, namely pterocarpans (glyceollins), isoflavones and coumestans. These prenylated isoflavonoids serve as defence compounds and can possess antimicrobial activity. Recently, we showed that priming with reactive oxygen species (ROS) specifically stimulated the production of glyceollins in Rhizopus spp.-elicited soybean seedlings (ROS + R). In this study, we achieved diversification of the inducible subclasses of prenylated isoflavonoids in soybean, by additional stimulation of two prenylated isoflavones and one prenylated coumestan. This was achieved by using a combination of the relatively long-lived ROS representative, H2O2, with AgNO3 prior to microbial elicitation. Microbial elicitation was performed with a live preparation of either a phytopathogenic fungus, Rhizopus spp. or a symbiotic bacterium, Bacillus subtilis. B. subtilis induced 30% more prenylated isoflavones than Rhizopus spp. in (H2O2 + AgNO3)-treated seedlings, without significantly compromising the total levels of glyceollins, compared to (ROS + R)-treated seedlings. The most abundant prenylated isoflavone induced was 6-prenyl daidzein, which constituted 60% of the total isoflavones. The prenylated coumestan, phaseol, was also induced in the (H2O2 + AgNO3)-treated and microbially elicited seedlings. Based on previously developed quantitative structure-activity relationship (QSAR) models, 6-prenyl daidzein and phaseol were predicted to be promising antibacterials. Overall, we show that treatment with H2O2 and AgNO3 prior to microbial elicitation leads to the production of promising antibacterial isoflavonoids from different subclasses. Extracts rich in prenylated isoflavonoids may potentially be applied as natural antimicrobial agents.

Soy-derived phytoalexins: mechanism of in vivo biological effectiveness in spite of their low bioavailability.

The well-demonstrated bioefficacy of phytochemicals in spite of their paradoxically low bioavailability has long puzzled scientists. Glyceollins, a family of soy-derived phytoalexins, have been reported to exert a variety of biological effects in vitro and in vivo systems in spite of poor systemic bioavailability after oral administration, suggesting that secondary messengers generated in gastrointestinal tract would transfer signals to target organs and tissues to manifest any effect. This review focuses on the potential mechanisms of how the poorly bioavailable glyceollins could still exert in vivo biological effects.

An Update on the Effects of Glyceollins on Human Health: Possible Anticancer Effects and Underlying Mechanisms.

Biologically active plant-based compounds, commonly referred to as phytochemicals, can influence the expression and function of various receptors and transcription factors or signaling pathways that play vital roles in cellular functions and are then involved in human health and diseases. Thus, phytochemicals may have a great potential to prevent and treat chronic diseases. Glyceollins, a group of phytoalexins that are isolated from soybeans, have attracted attention because they exert numerous effects on human functions and diseases, notably anticancer effects. In this review, we have presented an update on the effects of glyceollins in relation to their potential beneficial roles in human health. Despite a growing number of studies suggesting that this new family of phytochemicals can be involved in critical cellular pathways, such as estrogen receptor, protein kinase, and lipid kinase signaling pathways, future investigations will be needed to better understand their molecular mechanisms and their specific significance in biomedical applications.

Protective Effect of Glyceollins in a Mouse Model of Dextran Sulfate Sodium-Induced Colitis.

Glyceollins, which are derived from daidzein in soybean in response to various stimuli or stresses, have been reported to activate antioxidant/detoxifying enzymes in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent manner, in addition to exerting anti-inflammatory effects in murine macrophages. As the Nrf2 signaling pathway is known to antagonize nuclear factor (NF)-κB signaling, glyceollins likely have the potential to prevent or treat inflammatory bowel disease. Thus, this study was conducted to examine whether glyceollins could inhibit dextran sulfate sodium (DSS)-induced colitis in a mouse model. Ulcerative colitis (UC) was induced in male BALB/c mice by administering drinking water with 4% DSS for 5 days. Glyceollins (4 or 10 mg/kg of body weight) were orally administered 48 h before and after DSS treatment. We found that glyceollins alleviated histological colon damage and inflammation induced by DSS treatment. More specifically, glyceollins reduced plasma levels of inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, which were otherwise markedly increased by DSS treatment. Markers of tissue damage, including malondialdehyde and 8-hydroxy-2-guanosine, were significantly increased by DSS treatment; however, this effect was mitigated through concomitant treatment with glyceollins. Furthermore, nuclear accumulation of NF-κB p65 and the expression of inducible nitric oxide synthase were upregulated by glyceollins, consistent with the observed modulation of inflammatory markers. In conclusion, glyceollins have therapeutic potential for UC and merit further clinical study.

Differential abilities of Korean soybean varieties to biosynthesize glyceollins by biotic and abiotic elicitors.

Glyceollins synthesized in soybeans that are exposed to biotic or abiotic stress have been reported to have health benefits. Considering that glyceollins are de novo synthesized from daidzein via several enzymatic steps and that isoflavone concentration widely varies among soybean varieties, the abilities of 60 soybean cultivars to synthesize glyceollins were compared under different elicitation conditions. Soybeans accumulated glyceollins differentially depending upon the cultivar when elicited with Aspergillus sojae. Contrary to our hypothesis that high isoflavone varieties may accumulate glyceollins more efficiently upon elicitation, glyceollin accumulation in response to fungal elicitation was not related with the concentration of either total isoflavones or daidzein in soybeans. Rather the glyceollin levels were significantly affected by soybean cultivar and most effectively increased by fungal infection. The data suggest that the selection of a strong fungal elicitor and a soybean cultivar with genotype that highly expresses the genes involved in glyceollin biosynthesis is essential for efficient glyceollin production.

Suppression of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis by glyceollins.

SCOPE: Our previous study showed that glyceollins induced phase 2 detoxifying and antioxidant enzymes. As chemical carcinogens can be detoxified by metabolic activity of phase 2 enzymes, subsequently decreasing their tumorigenic potential, we investigated whether 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumorigenesis could be attenuated by treatment with glyceollins in a mouse model. METHODS AND RESULTS: Pretreatment with glyceollins (5 mg/kg body weight) caused a significant reduction in tumor formation and an increase in survival rate. The protective effect of glyceollins against DMBA-induced tumorigenesis was found to be mainly associated with their potential to induce phase 2/antioxidant enzymes that are, in turn, regulated by the nuclear factor E2-related factor 2 signaling pathway, and to a less extent to suppress phase 1 enzymes. As glyceollins increased the number of terminal end buds, a tumor-prone mammary tissue type in mice, it is unlikely that they exert their antitumorigenic action through their estrogenic activity. CONCLUSION: Glyceollins were found to have a protective effect in a chemically induced mammary tumor model.

Anti-septic effects of glyceollins in HMGB1-induced inflammatory responses in vitro and in vivo.

The ubiquitous nuclear protein High mobility group box 1 (HMGB1) is released by activated macrophages and human umbilical vein endothelial cells (HUVECs), and functions as a late mediator of experimental sepsis. Glyceollins (GCLs) are active compounds from Aspergillus sojae which have been reported for anti-cancer, anti-diabetes, and anti-inflammatory activities. We investigated here, the antiseptic effects and underlying mechanisms of GCLs against HMGB1-mediated septic responses in HUVECs and mice. According to the results, GCLs effectively inhibited lipopolysaccharide-induced release of HMGB1, and suppressed HMGB1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, GCLs suppressed the production of tumor necrosis factor-α and interleukin 6 and activation of nuclear factor-κB and extracellular regulated kinases 1/2 by HMGB1. Collectively, these results indicate that GCLs could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Inhibitory effect of glyceollins on vasculogenesis through suppression of endothelial progenitor cell function.

SCOPE: Endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells, and have the ability to differentiate into mature endothelial cells and contribute to neovascularization. Glyceollins are a type of phytoalexin produced in soybeans under stress conditions. The aim of this study is to determine the effect of glyceollin treatment on EPCs during early tumor vasculogenesis. METHODS AND RESULTS: We found that glyceollin treatment significantly decreased the number of EPC colony-forming units in human cord blood-derived AC133⁺ cells and mouse bone-marrow-derived c-Kit⁺/Sca-1⁺/Lin⁻ cells. Glyceollin treatment diminished the number of lineage-committed EPC cells in a dose-dependent manner (1-20 µM). Glyceollin treatment inhibited EPC migration, tube formation and the mRNA expression of angiopoietin-1 (Ang-1), Tie-2, stromal-derived factor-1 (SDF-1), C-X-C-chemokine receptor-4 (CXCR4), and endothelial nitric oxide synthase (eNOS) in cultured EPCs. Glyceollin treatment suppressed activation of Akt, Erk, and eNOS induced by SDF-1α or vascular endothelial growth factor (VEGF). Treatment with 10 mg/kg glyceollins significantly reduced the number of tumor-induced circulating EPCs and the incorporation of EPCs into neovessels in bone marrow transplanted mice. CONCLUSION: These results suggest that glyceollins inhibit the function of EPCs in tumor neovascularization. Glyceollins from soybean elicitation could be beneficial in prevention of cancer development via vasculogenesis.

Glyceollins, a novel class of soybean phytoalexins, inhibit SCF-induced melanogenesis through attenuation of SCF/c-kit downstream signaling pathways

The anti-melanogenesis effect of glyceollins was examined by melanin synthesis, tyrosinase activity assay in zebrafish embryos and in B16F10 melanoma cells. When developing zebrafish embryos were treated with glyceollins, pigmentation of the embryos, melanin synthesis and tyrosinase activity were all decreased compared with control zebrafish embryos. In situ expression of a pigment cell-specific gene, Sox10, was dramatically decreased by glyceollin treatment in the neural tubes of the trunk region of the embryos. Stem cell factor (SCF)/c-kit signaling pathways as well as expression of microphthalmia-associated transcription factor (MITF) were determined by western blot analysis. Glyceollins inhibited melanin synthesis, as well as the expression and activity of tyrosinase induced by SCF, in a dose-dependent manner in B16F10 melanoma cells. Pretreatment of B16F10 cells with glyceollins dose-dependently inhibited SCF-induced c-kit and Akt phosphorylation. Glyceollins significantly impaired the expression and activity of MITF. An additional inhibitory function of glyceollins was to effectively downregulate intracellular cyclic AMP levels stimulated by SCF in B16F10 cells. Glyceollins have a depigmentation/whitening activity in vitro and in vivo, and that this effect may be due to the inhibition of SCF-induced c-kit and tyrosinase activity through the blockade of downstream signaling pathway.