μM Genistein Research Articles

Cooperative effects of soy isoflavones and carotenoids on osteoclast formation

Osteoclasts play a major role in bone resorption. Several functional food components, such as soy isoflavones and carotenoids, are reported to inhibit osteoclast formation. However, the cooperative effect of functional foods or their constituents on bone metabolism has not been clarified. This study aimed to investigate the cooperative effect of soy isoflavones and carotenoids on osteoclast formation in vitro using cultures of RAW264 and bone marrow cells in the presence of receptor activator of nuclear factor κ-B ligand. In RAW264 cells, treatment with soy isoflavones (genistein or equol) or carotenoids (β-carotene) suppressed osteoclast formation. At 10 µM, genistein and equol inhibited RAW264 cell proliferation but did not affect cell viability. When 10 µM genistein or equol was combined with 0.1 µM β-carotene, we observed an additive suppressive effect on osteoclast differentiation. Similar results were observed with bone marrow cell cultures. We found that 10 µM of zeaxanthin or lutein suppressed osteoclast formation singly, and further enhanced the suppressive effects of daidzein or genistein when administered in combination. These results suggest that the combination of soy isoflavones and carotenoids have an enhanced suppressive effect on osteoclast formation. This knowledge might be important in planning diet for bone health.

Genistein Enhances the Radiosensitivity of Breast Cancer Cells via G2/M Cell Cycle Arrest and Apoptosis

The aim of the present study was to investigate the radiosensitizing effect of genistein, and the corresponding mechanisms of action on breast cancer cells with different estrogen receptor (ER) status. Human breast cancer cell lines such as MCF-7 (ER-positive, harboring wild-type p53) and MDA-MB-231 (ER-negative, harboring mutant p53) were irradiated with X-rays in the presence or absence of genistein. Cell survival, DNA damage and repair, cell cycle distribution, cell apoptosis, expression of proteins related to G2/M cell cycle checkpoint and apoptosis were measured with colony formation assays, immunohistochemistry, flow cytometry and western blot analysis, respectively. Genistein showed relatively weak toxicity to both cell lines at concentrations in the range of 5–20 μM. Using the dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at a 10% cell survival (IC10) were 1.43 for MCF-7 and 1.36 for MDA-MB-231 cells, respectively. Significantly increased DNA damages, arrested cells at G2/M phase, decreased homologous recombination repair protein Rad51 foci formation and enhanced apoptotic rates were observed in both cell lines treated by genistein combined with X-rays compared with the irradiation alone. The combined treatment obviously up-regulated the phosphorylation of ATM, Chk2, Cdc25c and Cdc2, leading to permanent G2/M phase arrest, and up-regulated Bax and p73, down-regulated Bcl-2, finally induced mitochondria-mediated apoptosis in both cell lines. These results suggest that genistein induces G2/M arrest by the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway and ultimately enhances the radiosensitivity of both ER+ and ER- breast cancer cells through a mitochondria-mediated apoptosis pathway.

Genistein Excitation of Gonadotrophin‐Releasing Hormone Neurones in Juvenile Female Mice

We investigated the effects of the phytoestrogen genistein on gonadotrophin-releasing hormone (GnRH) neurones using single-cell electrophysiology on GnRH-green fluorescent protein (GFP) transgenic juvenile female mice. Perforated patch-clamp recordings from GnRH-GFP neurones showed that approximately 83% of GnRH neurones responded to 30 μm genistein with a markedly prolonged membrane depolarisation. This effect not only persisted in the presence of tetrodotoxin, but also in the presence of amino acid receptor antagonists, indicating the direct site of action on postsynaptic GnRH neurones. Using a voltage clamp technique, we found that 30 μm genistein increased the frequency of synaptic current of GnRH neurones clamped at -60 mV in the presence of glutamate receptor blocker but not GABAA receptor blocker. Pre-incubation of GnRH neurones with 30 μm genistein enhanced kisspeptin-induced membrane depolarisation and firing. GnRH neurones of juvenile mice injected with genistein in vivo showed an enhanced kisspeptin response compared to vehicle-injected controls. The transient receptor potential channel (TRPC) blocker 2-aminoethoxydiphenyl borate (75 μm) blocked the genistein-mediated response on GnRH neurones. These results demonstrate that genistein acts on GnRH neurones in juvenile female mice to induce excitation via GABA neurotransmission and TRPCs to enhance kisspeptin-induced activation.

Agrobacterium rhizogenes transformed soybean roots differ in their nodulation and nitrogen fixation response to genistein and salt stress

We evaluated response differences of normal and transformed (so-called 'hairy') roots of soybean (Glycine max L. (Merr.), cv L17) to the Nod-factor inducing isoflavone genistein and salinity by quantifying growth, nodulation, nitrogen fixation and biochemical changes. Composite soybean plants were generated using Agrobacterium rhizogenes-mediated transformation of non-nodulating mutant nod139 (GmNFR5α minus) with complementing A. rhizogenes K599 carrying the wild-type GmNFR5α gene under control of the constitutive CaMV 35S promoter. We used genetic complementation for nodulation ability as only nodulated roots were scored. After hairy root emergence, primary roots were removed and composite plants were inoculated with Bradyrhizobium japonicum (strain CB1809) pre-induced with 10 μM genistein and watered with NaCl (0, 25, 50 and 100 mM). There were significant differences between hairy roots and natural roots in their responses to salt stress and genistein application. In addition, there were noticeable nodulation and nitrogen fixation differences. Composite plants had better growth, more root volume and chlorophyll as well as more nodules and higher nitrogenase activity (acetylene reduction) compared with natural roots. Decreased lipid peroxidation, proline accumulation and catalase/peroxidase activities were found in 'hairy' roots under salinity stress. Genistein significantly increased nodulation and nitrogen fixation and improved roots and shoot growth. Although genistein alleviated lipid peroxidation under salinity stress, it had no significant effect on the activity of antioxidant enzymes. In general, composite plants were more competitive in growth, nodulation and nitrogen fixation than normal non-transgenic even under salinity stress conditions.

Antiproliferative activity of daidzein and genistein may be related to ERα/c-erbB-2 expression in human breast cancer cells

In this study, we investigated the antiproliferative activity of the isoflavones daidzein and genistein in three breast cancer cell lines with different patterns of estrogen receptor (ER) and c‑erbB‑2 protein expression (ERα‑positive MCF‑7 cells, c‑erbB‑2‑positive SK‑BR‑3 cells and ERα/c‑erbB‑2‑positive ZR‑75‑1). After treatment at various concentrations (1‑200µM for 72h), the effect of daidzein and genistein on the proliferation of different cell types varied; these effects were found to be associated with ERα and c‑erbB‑2 expression. Daidzein and genistein exhibited biphasic effects (stimulatory or inhibitory) on proliferation and ERα expression in MCF‑7cells. Although 1µM daidzein significantly stimulated cell growth, ERα expression was unaffected. However, genistein showed marked increases in proliferation and ERα expression after exposure to <10µM genistein. Notably, the inhibition of cell proliferation by 200µM genistein was greater compared to that by daidzein at the same concentration. Daidzein and genistein significantly inhibited proliferation of SK‑BR‑3 and ZR‑75‑1 cells in a dose‑dependent manner. In addition, ERα and c‑erbB‑2 expression was reduced by daidzein and genistein in both SK‑BR‑3 and ZR‑75‑1 cells in a dose‑dependent manner. However, the effect of genistein was greater compared to that of daidzein. In conclusion, the isoflavones daidzein and genistein showed anti‑breast cancer activity, which was associated with expression of the ERα and c‑erbB‑2 receptors.

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

Formation of Phosphoglycosides in Caenorhabditis elegans: A Novel Biotransformation Pathway

Background Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition.Principal Findings C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide.Conclusions/SignificanceOur study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.

Genistein Suppresses Prostate Cancer Growth through Inhibition of Oncogenic MicroRNA-151

Genistein has been shown to suppress the growth of several cancers through modulation of various pathways. However, the effects of genistein on the regulation of oncogenic microRNA-151 (miR-151) have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer (PCa). Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 µM genistein down-regulated the expression of miR-151 compared with vehicle control. Inhibition of miR-151 in PCa cells by genistein significantly inhibited cell migration and invasion. In-silico analysis showed that several genes (CASZ1, IL1RAPL1, SOX17, N4BP1 and ARHGDIA) suggested to have tumor suppressive functions were target genes of miR-151. Luciferase reporter assays indicated that miR-151 directly binds to specific sites on the 3′UTR of target genes. Quantitative real-time PCR analysis showed that the mRNA expression levels of the five target genes in PC3 and DU145 were markedly changed with miR-151 mimics and inhibitor. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had an adverse effect on survival rate. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of PCa.

Abstract 2299: Genistein suppresses prostate cancer growth through inhibition of oncogenic miR-151

Abstract Introduction and Objective: Genistein has been shown to suppress the growth of several cancer cell lines in vitro and in vivo, reducing the expression of several oncogenic microRNAs. However, the effects of genistein on the regulation of oncogenic miR-151 have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer. Methods: The expression levels of miR-151 were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. Nineteen clinical specimens were obtained from patients at the San Francisco Veterans Affairs Medical Center from 2000 to 2006. For gain-and loss-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), and invasion (matrigel invasion assay) were evaluated in human prostate cancer cell lines, PC3 and DU145. Predicted target genes and their target microRNA binding site seed regions were investigated using TargetScan and microRNA.org. Genistein was administered every day along with a change of medium, and the cells were grown for four days. Results: Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 μM genistein down-regulated the expression of miR-151 compared with vehicle control. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had a significant adverse effect on survival rate. Inhibition of miR-151 in prostate cancer cells by genistein significantly inhibited cell migration and invasion. By in-cilico analysis, several genes (N4BP1, CASZ1, IL1RAPL1, LGI1, SOX17 and TOM1L1) that are suggested to have tumor suppressive function in several types of cancers were detected as candidate target genes of miR-151. Conclusions: We demonstrated that genistein down-regulated miR-151 that functions as an oncogenic miRNA in prostate cancer. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of prostate cancer. Source of Funding: Grants R01CA160079-01, R01CA138642, VA Research Enhancement Award Program (REAP), and VA Merit Review grants Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2299. doi:1538-7445.AM2012-2299

Abstract 4113: Combination of LC3 II shRNA and genistein completely inhibited rapamycin-induced autophagy and promoted apoptosis in human malignant neuroblastoma cell lines

Abstract Neuroblastoma is an extracranial solid tumor that mainly occurs in children. It is derived from embryonic neural crest cells of the peripheral sympathetic nervous system. The overall survival rate of malignant neuroblastoma patients is very poor despite multi-modal therapy including surgery, radiotherapy, and chemotherapy. Efficacy of chemotherapy is often compromised due to presence of autophagy, which is a survival mechanism in solid tumors. Autophagy is a catablolic process for lysosomal degradation of cytoplasmic contents for recycling and it is activated during stress such as nutrient starvation and growth factor deprivation. The hallmark of autophagy is generation of double membrane structure called autophagosome that contains the microtubule-associated protein light chain 3 form II (LC3 II). In this study, we used rapamycin to mimic starvation-induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR32 cells and then investigated capability of the combination of LC3 II knockdown and genistein (GST) treatment for inhibition of autophagy and promotion of apoptosis. First, we treated both cell lines with 200 nM rapamycin for 6, 12, and 24 h and performed acridine orange (AO) staining, flow cytometric analysis, RT-PCR, and Western blotting to establish time-dependent increases in autophagic features such as formation of acidic vesicular organelles (AVO) and LC3 II. Then, we carried out dose-response studies by transfecting the rapamycin-exposed cell lines with various concentrations of LC3 II short hairpin RNA (shRNA) plasmid and treating them with several doses of GST alone and in combination. MTT assays showed the highest synergistic efficacy in inhibiting cell viability using the combination of 50 nM LC3 II shRNA and 25 µM GST in SK-N-BE2 cells while the combination of 100 nM LC3 II shRNA and 25 µM GST in IMR32 cells. Our AO staining and flow cytometric quantitation of AVO confirmed that combination of LC3 II shRNA and GST completely inhibited rapamycin-induced autophagy to promote apoptotic death. In situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy most effectively induced apoptosis in both cell lines when compared with control shRNA, LC3 II shRNA, or GST alone. We unraveled the molecular mechanisms for inhibition of autopphagy and induction of apoptosis by Western blotting of the proteins involved in these processes. Down regulation of LC3 II and upregulation of mTOR in both cell lines showed inhibition of autophagy. Combination therapy promoted mitochondrial pathway leading to activation of caspase-3 and degradation of PARP for inducing apoptosis. Collectively, our results clearly show that combination of LC3 II knockdown and GST treatment serves as promising therapeutic strategy for inhibition of autophagy and induction of apoptosis in different human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4113. doi:1538-7445.AM2012-4113

Concentration-dependent effects of the soy phytoestrogen genistein on the proteome of cultured cardiomyocytes

The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1μM) and high (50μM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1μM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes.

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate

In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 μM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction.

Mechanism of Reactive Dilation in Soleus Feed Arteries

We have previously shown that reactive dilation of isolated resistance arteries is dependent on both magnitude and duration of pressure reduction. We sought to determine the mechanism for these differences. Rat soleus feed arteries (n=54) were isolated, cannulated, and intraluminal pressure was set at either 65 or 115 cmH2O. Intraluminal pressure was reduced to 14 cmH2O from either baseline pressure for 1 or 2 min and returned abruptly to baseline pressure, causing a temporary dilation lasting 1–2 minutes. Increasing either duration or baseline pressure increased the magnitude of dilation (1m‐65 = 22.9±2, 1m‐115 = 32.2±2.4, 2m‐65 = 29±2.1, 2m‐115 = 45.1±2.8 max % dilation). Inhibition of nitric oxide production (300 μM L‐NNA) or inhibition of mechanosensitive cation channels (50 μM gadolinium) both failed to alter reactive dilation. Likewise, inhibiting depolymerization of actin filaments (100 nM jasplakinolide) did not reduce reactive dilation. Finally, neither inhibition of tyrosine kinase activity (5 μM genistein) nor inhibition of large Ca2+ activated K+ channels (BKCa) (5 μM iberiotoxin) reduced reactive dilation. These data indicate that reactive dilation of soleus feed arteries is not dependent on nitric oxide, mechanosensitive cation channels, actin depolymerization, tyrosine‐kinase activity, or BKCa. The mechanism responsible for reactive dilation of soleus feed arteries remains unknown.

Comparative Proteomic Profiling of Human Breast Cell Lines

Although epidemiological studies have suggested that the isoflavone genistein found in soy may be protective against breast cancer, its effects on molecular processes involved in cell proliferation have not been clearly elucidated. Using comparative shotgun proteomics, we examined the proteome of non‐cancerous breast MCF10A cells, estrogen receptor‐positive breast cancer T47D cells, and Hras transfected MCF10A cells after treatment with 3, 10, and 30 μM genistein for 18 h. While genistein induced G1‐phase arrest in MCF10A‐Hras and G2/M‐phase arrest in T47D cells, it did not alter the cell cycle distribution in MCF10A cells. The arrest in G1‐phase of MCF10A‐Hras cells coincided with downregulation of the proliferation marker Ki67, epidermal growth factor receptor, various components of the mitogen‐activated protein kinase family of enzymes and low molecular weight phosphotyrosine protein phosphatase (ACP1), The arrest in G2/M‐phase of T47D cells was accompanied by upregulation of caspase‐8 and mSH2, suggesting that genistein may induce a G2/M checkpoint/proapoptotic response involving the mismatch repair system. Taken together, these findings suggest a differential response to genistein in cancerous cells and non‐malignant epithelial cells, and demonstrate the utility of comparative shotgun proteomics for investigating the cancer preventative effects of bioactive components.

Can Soy Phytoestrogens Decrease DNA Methylation in BRCA1 and BRCA2 Oncosuppressor Genes in Breast Cancer?

Although soy phytoestrogens have been postulated to exert a protective effect against breast cancer, the attendant mechanisms, in particular epigenetics underpinnings, have remained elusive. We investigated the putative effects on DNA methylation by two naturally occurring isoflavones, genistein and daidzein, in a study of the BRCA1 and BRCA2 oncosuppressor genes in breast cancer cell lines (MCF-7, MDA-MB 231, and MCF10a). A demethylant agent, the 5-azacytidine, and a methylant, the budesonide, were used as treatment controls. DNA methylation of BRCA1 and BRCA2 was investigated with methylated DNA immunoprecipitation coupled with PCR. In parallel, protein expression was determined by Western blot, immunohistochemistry, and confocal microscopy. Our results suggest that treatment with 18.5 μM Genistein or 78.5 μM Daidzein might reverse DNA hypermethylation and restore the expression of the oncosuppressor genes BRCA1 and BRCA2. 5-Azacitydine also enhanced the reexpression of these genes while budesonide had an opposite effect. To the best of our knowledge, these observations, while requiring replication, provide new evidence on potential epigenetic mechanisms by which genistein and daidzein might contribute to regulation of the BRCA1 and BRCA2. Future studies are warranted on whether the demethylating effect of genistein and daidzein is global or focused on select candidate genes.

(&amp;plusmn;)Equol inhibits invasion in prostate cancer DU145 cells possibly via down-regulation of matrix metalloproteinase-9, matrix metalloproteinase-2 and urokinase-type plasminogen activator by antioxidant activity

Exposure to soy isoflavones has been associated with low mortality of prostate cancer. In this study, we examined the effects of (±)equol and two representative isoflavones, daidzein and genistein, on migration and invasion in human prostate cancer DU145 cells. First of all, the three regents did not show significant growth inhibitive effect in DU145 cells until the treatments last for 72 h. Treatment with 5 µM, 10 µM, 50 µM (±)equol, 0.5 µM, 1 µM, 5 µM daidzein and genistein for 24 h decreased cell migration and invasion significantly. (±)equol activated phosphatase and tensin homologue deleted on chromosome ten at protein level but not mRNA level, which activated antioxidants, including superoxide dismutase and nuclear factor (erythroid-derived 2)-like 2. A reduction of malondialdehyde concentration, the product of lipid per-oxidation, was observed as well. Moreover, matrix metalloproteinase-2, matrix metalloproteinase-9, and urokinase-type plasminogen activator, the crucial members in metastasis, were down-regulated. Overall, our data indicate that (±)equol, daidzein and genistein may have significant anti-invasion effect in DU145 cells (in vitro). The effects induced by (±)equol may relate to its anti-oxidant effect mediated by phosphatase and tensin homologue deleted on chromosome ten.

Sensitization of Cervical Cancer Cells to Cisplatin by Genistein: The Role of NFB and Akt/mTOR Signaling Pathways

Cervical cancer is among the top causes of death from cancer in women. Cisplatin-based chemotherapy has been shown to improve survival; however, cisplatin treatment is associated with toxicity to healthy cells. Genistein has been used as an adjunct to chemotherapy to enhance the activity of chemotherapeutic agents without causing increased toxicity. The present study was designed to investigate the effect of genistein (25 μM) on antitumor activity of cisplatin (250 nM) on HeLa cervical cancer cells. We have examined the alterations in expression of NF-κB, p-mTOR, p-p70S6K1, p-4E-BP1, and p-Akt protein levels in response to treatment. The combination of 25 μM genistein with 250 nM cisplatin resulted in significantly greater growth inhibition (P < 0.01). Genistein enhanced the antitumor activity of cisplatin and reduced the expression of NF-κB, p-mTOR, p-p70S6K1, p-4E-BP1, and p-Akt. The results in the present study suggest that genistein could enhance the activity of cisplatin via inhibition of NF-κB and Akt/mTOR pathways. Genistein is a promising nontoxic nutritional agent that may enhance treatment outcome in cervical cancer patients when given concomitantly with cisplatin. Clinical trials of genistein and cisplatin combination are warranted to test this hypothesis.

Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 μM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ(EcfG) regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.

Depolarization Induces NR2A Tyrosine Phosphorylation and Neuronal Apoptosis

Cytosol Ca2+ overload plays a vital role in ischemic neuronal damage, which is largely contributed by the Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs) and N-methyl-D-aspartate (NMDA) type glutamate receptors. In this article, L-VGCCs were activated by depolarization to investigate the cross-talk between NMDA receptors and L-VGCCs. Depolarization was induced by 20 minutes incubation of 75 mM KCl in cultured rat cortical neuron. Apoptosis-like neuronal death was detected by DAPI staining. Tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A), interactions of Src and NR2A were detected by immunoblot and immunoprecipitation. Depolarization induced cortical neuron apoptosis-like cell death after 24 hours of restoration. The apoptosis was partially inhibited by 5 mM EGTA, 100 μM Cd2+, 10 μM nimodipine, 100 μM genistein, 20 μM MK-801, 2 μM PP2 and combined treatment of nimodipine and MK-801. NR2A tyrosine phosphorylation increased after depolarization, and the increase was inhibited by the drugs listed above. Moreover, non-receptor tyrosine kinase Src bound with NR2A after depolarization and restoration. The binding was also inhibited by the drugs listed above. The results indicated that depolarization-induced neuronal death might be due to extracellular Ca2+ influx through L-VGCCs and subsequently Src activationmediated NR2A tyrosine phosphorylation.

2B4 (CD244) is Involved in Eosinophil Adhesion and Chemotaxis, and its Surface Expression is Increased in Allergic Rhinitis after Challenge

A role for the subtypes of CD2 Ig superfamily receptors has been recently demonstrated in eosinophilic inflammation in experimental asthma and atopic asthmatics. We investigated the functions of 2B4 (CD244) molecules in eosinophil adhesion and chemotaxis, and correlated the results to the pathophysiology of allergic rhinitis (AR). Herein, we show that agonistic stimulation of 2B4 by C1.7, the anti-human 2B4 functional grade purified antibody, resulted in significant increase of eosinophils and eosinophil cell line (Eol-1 cells) adhesion to collagen type IV, and random migration. These functions were associated with tyrosine kinase phosphorylation of several protein residues of low molecular weight. Flow cytometry (FACS) experiments demonstrated that Eol-1 cells, normal peripheral blood eosinophils and eosinophils from AR patients, express surface 2B4 molecules. In vitro AR model demonstrated that the CC-chemokine receptor CCR3 stimulation by eotaxin induced significant increase in the expression of surface 2B4 in eosinophils and Eol-1 cells. Immunofluorescence confocal microscopy images showed that eotaxin induces also redistribution of 2B4 molecules towards the pseudopods in eosinophils and Eol-1 cells, changing their shape. Blocking of 2B4 molecules by the corresponding neutralizing antibody inhibited eotaxin induced Eol-1-adhesion, chemotaxis and the cytoskeleton changes. Pretreatment of Eol-1 cells with 1 microM genistein blocked eotaxin-induced Eol-1 adhesion, chemotaxis and 2B4 up-regulated expression. In vivo correlation demonstrated the expression of 2B4 molecules in eosinophils from AR patients to be significantly increased, after nasal provocation challenge. These results identify a novel role for 2B4 molecules in eosinophil functional migratory response and may point to a novel tyrosine kinase-mediated ligation between CCR3 receptor and 2B4 co-receptor in eosinophil chemotaxis. If so, then 2B4 molecules would be a novel target for therapeutic modalities in diseases characterized by eosinophilia such as AR.