Biotransformed soybean extract induces cell death of estrogen-dependent breast cancer cells by modulation of apoptotic proteins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells

Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60 μM) and soybean components genistein (Gen, 2 μM), lunasin (Lun, 2 μM), β-conglycinin (β-con, 3 μM), and glycinin (Gly, 3 μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET + Lun + Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC.

The effects of conjugated estrogen, raloxifene and soy extract on collagen in rat bones

Objective To evaluate the action of conjugated equine estrogen, raloxifene and isolated or combined genistein-rich soy extracts on collagen fibers in the bones of oophorectomized rats. Materials and methods Seventy female rats received testosterone propionate (0.1 mu g/g) on the 9th day after birth. At 6 months of age, the rats were administered the vehicle (propylene glycol, 0.5 ml/day), and ten of the rats were randomly chosen to comprise the non-oophorectomized control group (GI). The other 60 rats were ovariectomized and randomized into six groups of ten as follows: GII, vehicle; GIII, conjugated equine estrogen (CEE), 50 mu g/kg/day; GIV, raloxifene (RAL), 0.75 mg/kg/day; GV, genistein-rich soy extract (GSE), 300 mg/kg/day; GVI, CEE + GSE, 50 mu g/kg/day + 300 mg/kg/day; and GVII, CEE + RAL, 50 mu g/kg/day + 0.75 mg/kg/day. Three months after surgery, the drugs were administered for 60 consecutive days. All rats were euthanized, and their left tibiae were removed for histological routine. The histological sections were stained with hematoxylin-eosin, and picrosirius for evaluating bone microarchitecture. Types I and II collagen fibers were analyzed by immunofluorescence. Data analysis was carried out with ANOVA and Tukey's test. Results Collagen reduction was significant in the GIII animals when compared to the other groups (p < 0.05). There was no significant difference in the thickness of collagen fibers among the groups. There was a greater quantity of type III collagen in GVI than in the other groups. Conclusion Our data indicate that conjugated equine estrogen improves bone quality because it increases the quantity of type I collagen while reducing the quantity of thin collagen fibers. In addition, the combination of CEE and raloxifene or genistein-rich soy extract is not as efficient as CEE itself to improve bone quality.

Effects of phytoestrogens derived from soy bean on expression of adhesion molecules on HUVEC

Background The risks of hormone replacement therapy have led to a search for new alternatives such as phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soybean, red clover and other plants. Due to this estrogen-like activity, phytoestrogens can have some effect on atherosclerosis. Human umbilical vein endothelial cells (HUVEC) have been extensively used to study the biology and pathobiology of human endothelial cells and most of the knowledge acquired is due to experiments with cultures of these cells. Objective To evaluate the effects of the phytoestrogen extracts from Glycine max soy bean, genistein, formononetin, biochanin A and daidzein, as well as a mixture of these extracts (Mix), on expression of adhesion molecules, VCAM-1, ICAM-1 and E-selectin, by endothelial cell HUVEC, stimulated with lipopolysaccharide. Methods HUVEC were cultured in medium EBM2, pretreated with isoflavones for 24 and 48 h and then stimulated with lipopolysaccharide; in addition, isoflavones were added, after stimulation by lipopolysaccharide, to HUVEC. We evaluated the production of VCAM-1, ICAM-1 and E-selectin on cell surface, by cell-based enzyme immunoassay, and of sVCAM-1, sICAM-1 and sE-selectin in culture supernatant, by ELISA. Results Genistein, formononetin, biochanin A and daidzein, as well as the Mix were able to reduce VCAM-1, ICAM-1 and E-selectin on cell surface and in culture supernatant. Conclusion Isoflavones extracted from Glycine max soy bean, in vitro, presented antiatherogenic effects, reducing the expression of adhesion molecules and acting as preventive agents as well as therapeutic agents.

Thermal behavior of malonylglucoside isoflavones in soybean flour analyzed by RPHPLC/DAD and eletrospray ionization mass spectrometry

Soybean (Glycine max (Merrill) L) contains high content of aglycone isoflavones, as well as glucoside and malonylconjugates. In this work, the content of isoflavones in defatted soy flour was determined by reversed-phase high-performance liquid chromatography (RPHPLC) after alcoholic extraction in methanol/water mixture in the ratio 80:20 (v/v). It was observed that the heating treatment transformed the malonylglucosides into glucoside isoflavones. After heat treatment at 121 degrees C for 30 min, nearly all malonylisoflavones were converted into glucoside, but acetylisoflavones were not detected via RPHPLC analysis. Electrospray ionization mass spectrometry confirmed the presence of malonylisoflavones in heat-treated defatted soy flour by direct infusion analysis. (c) 2012 Elsevier Ltd. All rights reserved.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

Secondary metabolites from Spirotropis longifolia (DC) Baill and their antifungal activity against human pathogenic fungi

A phytochemical study of the ethyl acetate extract of the roots and adventitious roots of Spirotropis longifolia, a monodominant tree species of the Guianan rainforest, has allowed the isolation of three compounds: 2- hydroxy-8,9-methylenedioxy-2',2'-dimethylpyrano-[5',6':4,3]-6a-prenyl-[6aS,11aS]-pterocarpan (spirotropin A), 2-hydroxy-8,9-methylenedioxy-2',2'-dimethy1-3',4'-dihydropyrano-[5',6':4,3]-6a-prenyl-(6aS,11aS]-pterocarpan (spirotropin B), and 5,7-dihydroxy-6.8-dipreny1-2 ''''.2 ''''-dimethylpyrano[5 '''',6 '''': 3',4]-isoflavone (spirotropone). In addition, 10 known compounds, trans-oxyresveratrol, trans-resveratrol, piceatannol, daidzein, genistein, isoprunetin, lupeol, latifolol, gnetin D and gnetin E, were also isolated. These compounds were evaluated for their antifungal activity and their cytotoxicity, and their structures were established by 1D and 2D NMR, HRMS, CD and optical rotation measurements. (C) 2011 Elsevier Ltd. All rights reserved.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Agents used for chemoprevention of prostate cancer may influence PSA secretion independently of cell growth in the LNCaP model of human prostate cancer progression

BACKGROUND: The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform. METHODS: LNCaP and C4-2 cells were incubated with genistein, seleno-L-methionine, lycopene, DL-alpha-tocopherol, and trans-beta-carotene at three different concentrations and cell growth was determined by the MTT assay. PSA mRNA expression was assessed by quantitative real-time RT-PCR and secreted PSA protein levels were quantified by the microparticle enzyme immunoassay. RESULTS: Genistein, seleno-l-methionine and lycopene inhibited LNCaP cell growth, and the proliferation of C4-2 cells was suppressed by seleno-L-methionine and lycopene. PSA mRNA expression was downregulated by genistein in LNCaP but not C4-2 cells. No other compound tested altered PSA mRNA expression. PSA protein expression was downregulated by genistein, seleno-L-methionine, DL-alpha-tocopherol in LNCaP cells. In C4-2 cells only genistein significantly reduced the secretion of PSA protein. CONCLUSIONS: In the LNCaP progression model PSA expression depends on the compound, its concentration and on the hormonal dependence of the cell line used and does not necessarily reflect cell growth or death. Before potential substances are evaluated in clinical trials using PSA as a surrogate end point marker, their effect on PSA mRNA and protein expression has to be considered to correctly assess treatment response by PSA.

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells

Although hormone therapy with antiandrogens has been widely used for the treatment of prostate cancer, some antiandrogens may act as androgen receptor (AR) agonists that may result in antiandrogen withdrawal syndrome. The molecular mechanism of this agonist response, however, remains unclear. Using mammalian two-hybrid assay, we report that antiandrogens, hydroxyflutamide, bicalutamide (casodex), cyproterone acetate, and RU58841, and other compounds such as genistein and RU486, can promote the interaction between AR and its coactivator, ARA70, in a dose-dependent manner. The chloramphenicol acetyltransferase assay further demonstrates that these antiandrogens and related compounds significantly enhance the AR transcriptional activity by cotransfection of AR and ARA70 in a 1:3 ratio into human prostate cancer DU145 cells. Our results suggest that the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells. These findings may provide a good model to develop better antiandrogens without agonist activity.

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.