Inhibitory Effect Of Genistein Research Articles

The anti-cancer impact of genistein against acute lymphoblastic leukaemia by controlling DICER and AGO2 involved in cytoplasmic microRNAs biogenesis—a possible new clue to mode of action of genistein

IntroductionChildhood acute lymphoblastic leukaemia (ALL) is one of the most prevalent malignancies. MethodsThe alteration in expression of the ibonuclease III is a Protein Coding gene (DICER) and Argonaute RISC Catalytic Component 2 (AGO2) was assessed in human samples and four ALL cell lines. Then, the inhibitory effect of genistein on ALL cell lines was evaluated. ResultsUp-regulation of DICER and down-regulation of AGO2 were observed in 40 patients with ALL compared to 35 healthy controls. The comparison of their alteration among different stages revealed their role in the progression of ALL. Genistein had dose- and time-dependent anti-proliferative effects against ALL cancer cells through inducing apoptosis and decreasing the growth rate of malignant cells. Genistein significantly elevated the level of DICER, particularly in two cells (Molt-17 and Nalm-6). The up regulation of AGO2 was significant after genistein treatment in the four cell lines as compared to non-treated cells. ConclusionsAlteration in the expression of these genes is correlated with the progression of the disease. The alteration of AGO2 and DICER expressions in B-ALL cell lines after genistein treatment provide this evidence to support our hypothesis that genistein can inhibit the growth of cancerous cells, particularly ALL cells, by mediating changes to elements that contribute to the biogenesis of endogenous microRNAs, such as AGO2 and DICER, thus affecting different cellular pathways in cancerous cells. This study seems to have clarified another anti-cancerous mechanism of genistein, which can be promising.

Molecular docking analysis reveals the functional inhibitory effect of Genistein and Quercetin on TMPRSS2: SARS-COV-2 cell entry facilitator spike protein

BackgroundThe Transmembrane Serine Protease 2 (TMPRSS2) of human cell plays a significant role in proteolytic cleavage of SARS-Cov-2 coronavirus spike protein and subsequent priming to the receptor ACE2. Approaching TMPRSS2 as a therapeutic target for the inhibition of SARS-Cov-2 infection is highly promising. Hence, in the present study, we docked the binding efficacy of ten naturally available phyto compounds with known anti-viral potential with TMPRSS2. The aim is to identify the best phyto compound with a high functional affinity towards the active site of the TMPRSS2 with the aid of two different docking software. Molecular Dynamic Simulations were performed to analyse the conformational space of the binding pocket of the target protein with selected molecules.ResultsDocking analysis using PyRx version 0.8 along with AutoDockVina reveals that among the screened phyto compounds, Genistein shows the maximum binding affinity towards the hydrophobic substrate-binding site of TMPRSS2 with three hydrogen bonds interaction ( − 7.5 kcal/mol). On the other hand, molecular docking analysis using Schrodinger identified Quercetin as the most potent phyto compound with a maximum binding affinity towards the hydrophilic catalytic site of TMPRSS2 ( − 7.847 kcal/mol) with three hydrogen bonds interaction. The molecular dynamics simulation reveals that the Quercetin-TMPRSS complex is stable until 50 ns and forms stable interaction with the protein ( − 22.37 kcal/mol of MM-PBSA binding free energy). Genistein creates a weak interaction with the loop residues and hence has an unstable binding and exits from the binding pocket.ConclusionThe compounds, Quercetin and Genistein, can inhibit the TMPRSS2 guided priming of the spike protein. The compounds could reduce the interaction of the host cell with the type I transmembrane glycoprotein to prevent the entry of the virus. The critical finding is that compared to Genistein, Quercetin exhibits higher binding affinity with the catalytic unit of TMPRSS2 and forms a stable complex with the target. Thus, enhancing our innate immunity by consuming foods rich in Quercetin and Genistein or developing a novel drug in the combination of Quercetin and Genistein could be the brilliant choices to prevent SARS-Cov-2 infection when we consider the present chaos associated with vaccines and anti-viral medicines.

Genistein, a Natural Isoflavone, Alleviates Seizure-Induced Respiratory Arrest in DBA/1 Mice.

Objective: Sudden unexpected death in epilepsy (SUDEP) is a fatal event that ranks second in years of potential life lost among neurological disorders. Seizure-induced respiratory arrest (S-IRA) is the primary instigator leading to death in many SUDEP cases. However, there are currently no effective preventive strategies against S-IRA other than the seizure control. Therefore, it is critical to develop new avenues to prevent SUDEP by investigating the pharmacological interventions of S-IRA. In the present study, we examined the effect of genistein, an isoflavone found in various dietary vegetables, on the incidence of S-IRA in DBA/1 mice.Methods: DBA/1 mice exhibited generalized seizures and S-IRA when subjected to acoustic stimulation. Genistein was intraperitoneally administered alone or in combination with an adrenoceptor antagonist and a serotonin (5-HT) receptor antagonist, respectively. The effects of drug treatments on S-IRA incidence and seizure behaviors were examined.Results: The incidence of S-IRA in DBA/1 mice was significantly reduced 2 h after injection of genistein at 1–90 mg/kg as compared with that in the vehicle control. Genistein could block S-IRA without interfering with any component of seizures, especially at relatively lower dosages. The S-IRA-suppressing effect of genistein was reversed by an α2 adrenoceptor antagonist but was not altered by an α1 antagonist. The inhibitory effect of genistein on S-IRA was not affected by a 5-HT3 or 5-HT2A receptor antagonist.Significance: Our data show that genistein reduces S-IRA incidence and can specifically block S-IRA in DBA/1 mice. Its suppressing effect on S-IRA is dependent on activating α2 adrenoceptors. Our study suggests that genistein, a dietary supplement, is potentially useful to prevent SUDEP in at-risk patients.

Genistein suppresses ox-LDL-elicited oxidative stress and senescence in HUVECs through the SIRT1-p66shc-Foxo3a pathways.

The anti-senescence function of genistein is related to inhibiting oxidative stress, however, the mechanism has not been clarified. The present study aimed to explore the effects of genistein on oxidized low-density lipoprotein (ox-LDL)-induced endothelial senescence and the role of the sirtuin-1 (SIRT1)-66-kDa Src homology 2 domain-containing protein (p66Shc)-forkhead box protein O3 (Foxo3a) pathways in the process. In this paper, human umbilical vein endothelial cells were pretreated with 1000 nM genistein for 30 min and then incubated with 50 mg/L ox-LDL for another 12 h; meanwhile, the functions of adenovirus-mediated overexpression of p66shc and small interfering RNA-mediated silencing of SIRT1 were investigated. Results showed that genistein pretreatment alleviated ox-LDL-induced mitochondrial reactive oxygen species, the levels of oxidatively modified DNA (8-OHdG) and pai-1, and the activity of SA-β-gal, which was associated with mitigating p66shc. Further studies indicated the inhibitory effect of genistein on p66shc was correlated with suppressing the acetylation and phosphorylation of p66shc, and ameliorating its mitochondrial translocation by activating SIRT1. Moreover, the inactivated p66shc could enhance the activity of Foxo3a via restraining the phosphorylation and triggering nucleus accumulation. The study demonstrates genistein could prevent ox-LDL-induced mitochondrial oxidative stress and senescence through the SIRT1-p66shc-Foxo3a pathways.

Local administration of genistein as a local anesthetic agent inhibits the trigeminal nociceptive neuronal activity in rats

A modulatory role has been reported for the isoflavone, genistein, on voltage-gated Na+ channels in the trigeminal ganglion in vitro. However, the acute effects of genistein in vivo, particularly on nociceptive transmission in the trigeminal system, remain to be determined. The aim of the present study was to examine whether acute local genistein administration to rats attenuates the excitability of wide-dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo.Extracellular single unit recordings were made from SpVc WDR neurons in response to orofacial non-noxious and noxious mechanical stimulation of pentobarbital-anesthetized rats. The effects of local administration of genistein, lidocaine, and lidocaine with genistein to the receptive field on the discharge frequency of SpVc neurons were evaluated. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was significantly and dose-dependently (0.1−10 mM) inhibited by genistein, and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 10 min. The inhibitory effect of genistein lasted for 20 min and was reversible. No significant difference was seen between the relative magnitude of inhibition by genistein on the SpVc WDR neuronal discharge frequency for noxious and non-noxious stimulation. The mean magnitude of inhibition by genistein (10 mM) on SpVc neuronal discharge frequency was almost equal to that of the local anesthetic, 1 % lidocaine (37 mM). Local injection of half-dose of lidocaine replaced the half-dose of genistein. These results suggest that local injection of genistein into the peripheral receptive field suppresses the excitability of SpVc neurons, possibly via inhibition of voltage-gated Na+ channels in the nociceptive nerve terminals of trigeminal ganglion. Therefore, administration of genistein as a local anesthetic may provide relief from trigeminal nociceptive pain without side effects, thus contributing to the area of complementary and alternative medicines.

Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human CaV3.3 Channel.

Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, CaV3.1, CaV3.2, and CaV3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human CaV3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the CaV3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the CaV3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the CaV3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of in vitro and in silico techniques, we have identified interacting determinants relevant to the CaV3.3/genistein complex formation and propose that genistein directly blocks the function of the human CaV3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rational development of improved and novel low-voltage activated T-type calcium channel inhibitors.

Functional analysis of isoflavones using patient-derived human colonic organoids

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn’s disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.

Inhibitory Effects of Genistein on Vascular Smooth Muscle Cell Proliferation Induced by Ox-LDL: Role of BKCa Channels.

Background Oxidized low-density lipoprotein (Ox-LDL) is a crucial pathogenic factor for vascular diseases, which can induce the proliferation of vascular smooth muscle cells (VSMCs). Genistein is the main component of soybean isoflavone. Genistein has a variety of pharmacological properties in the treatment of vascular diseases and a promising clinical application. Large-conductance calcium-activated potassium (BKCa) channels are the primary type of potassium channels in VSMCs, which regulate various biological functions of VSMCs. However, whether genistein exerts an antiproliferation effect on Ox-LDL-stimulated VSMCs remains unclear. The current study is aimed at elucidating the effect of genistein on the Ox-LDL-stimulated proliferation of VSMCs and its possible molecular mechanism, especially the electrophysiological mechanism related to BKCa channels. Methods Monoculture VSMC was obtained by an acute enzyme-dispersing method. The proliferation of cells was measured by CCK-8, cell cycle, and proliferating cell nuclear antigen (PCNA) expression. The BKCa whole-cell currents were measured by patch-clamp. Results Ox-LDL treatment induced the proliferation of VSMCs, upregulated the BKCa protein expression, and increased the density of BKCa currents, while genistein significantly inhibited these effects caused by Ox-LDL. BKCa channels exerted a regulatory role in the proliferation of VSMCs in response to Ox-LDL. The inhibition of BKCa channels suppressed Ox-LDL-stimulated VSMC proliferation, while the activation of BKCa channels showed the opposite effect. Moreover, genistein suppressed the activity of BKCa, including protein expression and current density in a protein tyrosine kinase- (PTK-) dependent manner. Conclusion This study demonstrated that genistein inhibited the Ox-LDL-mediated proliferation of VSMCs by blocking the cell cycle progression; the possible molecular mechanism may be related to PTK-dependent suppression of BKCa channels. Our results provided novel ideas for the application of genistein in the treatment of vascular diseases and proposed a unique insight into the antiproliferative molecular mechanism of genistein.

HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells

Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3β signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.

Inhibitory effect of genistein and PTP1B on grasshopper Oedaleus asiaticus development

Protein tyrosine kinase (PTKs) and protein tyrosine phosphatase (PTPs) collectively regulate the insect insulin-signaling pathway (ISP), cell growth, metabolism, proliferation, differentiation, cell communication and monitoring of immune responses. We studied the inhibitory response of the grasshopper Oedaleus asiaticus Bey-Bienko to treat with the flavonoid genistein (PTK inhibitor), the enzyme PTP1B-IN-1 (PTP1B inhibitor) and control (dimethyl sulfoxide (DMSO)). Mean survival rates were not significantly different amongst the three treatments, but growth rate, body mass, and overall biological performance were reduced significantly following treatment with genistein. On comparison, treatment with PTP1B-IN-1 was not significantly different from the control. Relative gene expression of INSR, IRS, PI3K, PDK, and Akt was substantially lower (P < 0.05) following treatment with genistein compared to PTP1B-IN-1 and control. Similarly, gene expression and protein phosphorylation levels of the PTK5, PTP1B → FOXO cascade, indicators of stress response in the ISP, were also reduced in response to genistein treatments. The activity of the protective enzymes (POD and CAT) was upregulated by FOXO in response to PTP1B-IN-1. Our results demonstrated that genistein negatively regulates the ISP pathway, with a consequent effect on growth and development of O. asiaticus, as higher energy was required for detoxification. Given the negative effect of genistein on O. asiaticus physiology and development, the compound could be used in a biopesticide formulation for insect pest control in grasslands and other crop plants as a potential application to grasshopper biological control.

Genistein Decreases APP/tau Phosphorylation and Ameliorates Aβ Overproduction Through Inhibiting CIP2A.

Upregulation of Cancerous Inhibitor of PP2A (CIP2A) plays an important role in disease-related phosphorylation of tau/APP and tau pathology/Aβ overproduction through inhibiting PP2A in AD brain. Genistein has been shown to potently reduce CIP2A in experimental cancer treatment research. Whether Genistein can ameliorate AD pathology through targeting CIP2A needs further investigation. The inhibitory effects of Genistein on tau/APP phosphorylation and Aβ overproduction in AD cell models have been explored. HEK293-T cells were co-transfected with CIP2A and APP plasmids, or CIP2A and tau plasmids, with Genistein incubation at 0, 30, 60 or 120 µM for 48 h, cell viability and PP2A activities were measured. HEK293-T cells with CIP2A/APP overexpression treated with Genistein at 30 µM for 48 h were collected and lyzed for Western blotting detection of CIP2A, PP2Ac, APP-T668, total APP, PS1, BACE1, sAPPα and sAPPβ. Aβ40 and Aβ42 levels in cell supernatant, soluble fraction (RIPA) and insoluble fraction (formic acid soluble) of cell lysates were measured by ELISA. HEK293-T cells with CIP2A/tau overexpression treated with Genistein at 30 µM for 48 h were collected for Western blotting detection of CIP2A, PP2Ac, tau-S396, tau-S404 and total tau. Genistein effectively reduced CIP2A expression, and restored PP2A activities both in CIP2A/APP, CIP2A/tau co-expressed cells. Genistein reduced APP phosphorylation at T668 site and inhibited Aβ production. Meantime, Genistein ameliorated tau hyperphosphorylation through repressing the inhibitory effect of CIP2A on PP2A. CIP2A is a target of Genistein in AD therapy. Genistein reduces APP/tau hyperphosphorylation and Aβ production through inhibiting the effect of CIP2A on PP2A.

Inhibitory effects of genistein in combination with gefitinib on the hepatocellular carcinoma Hep3B cell line.

Combination therapy is an important method for treating advanced hepatocellular carcinoma (HCC). Gefitinib is an epidermal growth factor receptor (EGFR) inhibitor, which has profound effects on HCC. The purpose of the present study was to investigate the effects of genistein in combination with gefitinib on the proliferation and apoptosis of HCC cells and the associated mechanism. Cell counting kit-8 assay was performed to calculate the IC50 values and cytotoxicity, whilst flow cytometry was used to assess cell apoptosis. Protein expression was detected using western blot analysis. The IC50 of genistein and gefitinib on Hep3B cells were calculated to be 128.078 and 13.657 µM, respectively. Genistein in combination with gefitinib significantly inhibited cell viability, promoted apoptosis and reduced EGFR, vascular endothelial growth factor receptor and platelet-derived growth factor receptor phosphorylation. Genistein in combination with gefitinib promoted the expression of cleaved caspase-3 and cleaved poly ADP-ribose polymerase. In addition, combined treatment of genistein and gefitinib strongly inhibited the activation of the Akt/Erk/mTOR signaling pathway. In conclusion, findings from the present study suggest that genistein in combination with gefitinib inhibit HCC cell proliferation and promote apoptosis by inhibiting the Akt/Erk/mTOR pathway.

Genistein suppresses psoriasis-related inflammation through a STAT3–NF-κB-dependent mechanism in keratinocytes

Psoriasis is a chronic recurrent skin inflammatory disease, and inhibition of inflammation may be an effective means of treating psoriasis. The flavonoid genistein has a clear anti-inflammatory effect. However, the anti-psoriatic effects of genistein and their underlying mechanisms remain unclear. In this study, we investigated the effects of genistein on imiquimod (IMQ)-induced psoriasis-like skin lesions in vivo and explored the mechanisms underlying those effects in vitro. It was found that genistein can significantly improve IMQ-induced pathological scores of cutaneous skin lesions in mice, reduce epidermal thickness, and inhibit the expression of inflammatory factors，including interleukin (IL)-1β, IL-6, tumour necrosis factor-alpha (TNF-α), chemokine ligand 2 (CCL2), IL-17 and IL-23. In vitro studies, genistein inhibited the proliferation of human keratinocyte HaCaT cells and inhibited the expression of inflammatory factors in a dose-dependent manner which induced by TNFα. Further researches showed that genistein could also significantly inhibit phosphorylated STAT3 (pSAT3) expression in IMQ mice dorsal skin and in TNF-α-induced HaCaT cells. The inhibitory effect of genistein on the expression of IL-6, IL-23 and TNF-α was weakened after Stat3 siRNA in HaCaT cells. Genistein could also significantly inhibit TNF-α induced the nuclear translocation of NF-κB, and inhibit the phosphorylation of I-kBα (pI-kBα). After combining with NF-κB blocker BAY 11–7082, the effect of genistein down-regulate the expression of TNF-α and VEGFA was attenuated in HaCaT cells. The results suggest that genistein may be developed for the treatment of psoriasis lesions.

Lack of combination effects of soy isoflavones and taxane chemotherapy of castration-resistant prostate cancer.

Patients with cancer, including prostate cancer, often use dietary supplements, such as soy or isoflavones, before, during, or after therapy. There is little information about possible interactions between supplements and cancer chemotherapy. There are some reports suggesting enhancement by genistein of taxane chemotherapy for castrate-resistant prostate cancer (CRPC). We investigated whether physiologically attainable concentrations of soy isoflavones (≤10 μM) interact with taxanes on growth inhibition of CRPC cells in vitro and in vivo in nude mice exposed via the diet, on microtubule disassembly in vitro, and on P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. Genistein, daidzein, and equol did not affect growth of VCaP, 22Rv1, C4-2, and PC-3 CRPC cells or growth inhibition of these cells by docetaxel and cabazitaxel. These isoflavones did not inhibit microtubule disassembly in vitro or inhibit the microtubule effects of taxanes and genistein did not bind substantially to microtubules. Genistein considerably inhibited P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. However, dietary supplementation with genistein at 250 and 500 ppm did not affect the tumor growth inhibiting effect of docetaxel on 22Rv1 cells xenografted in nude mice. Our results with relevant cell models and clinically achievable concentrations of soy isoflavones do not support the notion that genistein or other soy isoflavones can enhance the effects of taxane chemotherapy in CRPC cell and xenograft models. Yet, the inhibitory effects of genistein on drug efflux in 22Rv1 cells and on microsomal CYP3A4 activity raise the possibility that genistein can affect taxane effects on CRPC cells in other circumstances than those we studied, which merits further research.

G protein-coupled estrogen receptor is involved in the anti-inflammatory effects of genistein in microglia

BackgroundGenistein (GEN), a phytoestrogen that is extracted from leguminous plants, can bind to estrogen receptor and exert biological effects. G protein-coupled estrogen receptor (GPER), a novel membrane estrogen receptor, has been reported to be involved in the anti-inflammatory process. In the present study, using BV2 microglial cell line and primary microglial culture, we evaluated the involvement of GPER in the anti-inflammatory effects of genistein against lipopolysaccharide (LPS)-induced microglia activation. MethodsThe anti-inflammatory effects of genistein were investigated in LPS-induced microglial activation in murine BV2 microglial cell line and primary microglial culture. Anti-inflammatory properties of genistein were determined by MTT, real time PCR, ELISA and western blot analysis. The pharmacological blockade and lentivirus-mediated siRNA knockdown of GPER were used to study the underlying mechanism. ResultsThe results showed that genistein exerted inhibitory effects on LPS-induced expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β) and interleukin-6 (IL-6). Pre-treatment with GPER antagonist G15 could significantly block the anti-inflammatory effects of genistein. Moreover, the inhibitory effects of genistein on LPS-induced activation of MAPKs and NF-κB signaling pathways could also be blocked by G15. Lentivirus-mediated siRNA knockdown of GPER significantly inhibited the anti-inflammatory effects of genistein in BV2 cells. Further study revealed that genistein treatment could increase the gene and protein expressions of GPER in BV2 cells. ConclusionTaken together, these data provide the first evidence that genistein exerts anti-inflammatory effects in microglial cells via GPER activation. These beneficial effects of genistein may represent a new strategy for the treatment of neuroinflammatory diseases.

Inhibitory effects of Genistein on hyperoxia-induced retinal neovascularization

Objective To investigate the effect of Genistein, a tyrosinase inhibitor, on retinal neovascularization in mice. Methods Thirty-six 7-day-old C57BL/6J mice were randomly assigned into hyperoxia-induced group, Genistein group, DMSO group and normal control group.The mice in the hyperoxia-induced group, Genistein group and DMSO group were fed in a static chamber with the oxygen volume fraction (75±2)% for 5 days and then sent back to natural environment for 5 days to establish retinal neovascular models, and 1 μl Genistein diluted by 5% dimethyl sulfoxide (DMSO) (400 mg/L) and 1 μl DMSO were intravitreally injected in the 12-day-old mice of Genistein group and DMSO group, respectively.The mice in the normal control group were bred in natural environment.The fluorescence angiography was carried out in 17-day-old mice (2 mice in each group) to prepare the whole retinal flatmounts, and the morphology of retinal vessels was observed under the fluorescence microscope.The other mice in various groups were sacrificed and the retinas were collected.The expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in mRNA and protein levels were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The use and care of the mice complied with regulations for the management of laboratory animals. Results Retinal vessels were normal in the mice of normal control group.In the mice of the hyperoxia-induced group and DMSO group, retinal vessels were tortuous, and neovacularization and non-perfusion areas were visible.In the Genistein group, retinal vessels were clearly visible, but non-perfusion areas were exhibited.The relative expression levels of VEGF mRNA in retinas were 0.64±0.25, 0.37±0.23, 0.03±0.02 and 0.04±0.02, and the relative expression levels of bFGF mRNA in retinas were 21.40±3.07, 17.22±2.63, 0.52±0.25 and 0.67±0.23, in the hyperoxia-induced group, DMSO group, Genistein group and normal control group, and the expressions of VEGF and bFGF in mRNA level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P<0.05). The protein expression levels of VEGF and bFGF were 1.01±0.05 and 0.97±0.06 in the hyperoxia-induced group, 1.06±0.07 and 1.03±0.08 in the DMSO group, 0.73±0.05 and 0.76±0.07 in the Genistein group, 0.52±0.05 and 0.56±0.05 in the normal control group.The expressions of VEGF and bFGF in protein level in the hyperoxia-induced group and DMSO group were significantly higher than those in the Genistein group and normal control group (all at P<0.05). Conclusions Genistein can inhibit hyperoxia-induced retinal neovascularization may be by down-regulating the expressions of VEGF and bFGF in retinas. Key words: Angiogenesis inhibitors/pharmacology; Neovascularization, retinal; hyperoxia; Vascular endothelial growth factor; Fibroblast growth factor

Inhibitory effect of genistein on MMP-2 and MMP-9 expression through suppressing NF-κB activity in peritoneum of murine model of endometriosis

Objective: To analyze the inhibitory effect of genistein on matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) through inhibition of nuclear factor-kappa B (NF-κB) activation. Methods: A total of 36 female mice were divided into six groups (n=6 in each group): control group (untreated mice), endometriosis mice group, and endometriosis mice groups administered with genistein at different doses (1.30 mg/day; 1.95 mg/day; 2.60 mg/day; and 3.25 mg/day). The genistein treatment was performed for 14 d. The expressions of NF-κB, MMP-2 and MMP-9 on the endometriosis lesions were analyzed by the immunohistochemical technique. Results: The activity of NF-κB in the endometriosis group increased significantly than that of the control group (P 0.05). The four doses of genistein administration were capable of significantly reducing the expression of MMP-2 compared to that of the endometriosis group (P 0.05). Administration of genistein at different doses was capable of significantly reducing the expression of MMP-9 compared to that of the endometriosis group (P 0.05). Conclusions: Genistein suppresses the expression of MMP-2 and MMP-9 through suppressing NF-κB activity in the peritoneum of murine model of endometriosis.

Genistein suppresses aerobic glycolysis and induces hepatocellular carcinoma cell death

Background:Genistein is a natural isoflavone with many health benefits, including antitumour effects. Increased hypoxia-inducible factor 1 α (HIF-1α) levels and glycolysis in tumour cells are associated with an increased risk of mortality, cancer progression, and resistance to therapy. However, the effect of genistein on HIF-1α and glycolysis in hepatocellular carcinoma (HCC) is still unclear.Methods:Cell viability, apoptosis rate, lactate production, and glucose uptake were measured in HCC cell lines with genistein incubation. Lentivirus-expressed glucose transporter 1 (GLUT1) or/and hexokinase 2 (HK2) and siRNA of HIF-1α were used to test the direct target of genistein. Subcutaneous xenograft mouse models were used to measure in vivo efficacy of genistein and its combination with sorafenib.Results:Genistein inhibited aerobic glycolysis and induced mitochondrial apoptosis in HCC cells. Neither inhibitors nor overexpression of HK2 or GLUTs enhance or alleviate this effect. Although stabiliser of HIF-1α reversed the effect of genistein, genistein no longer has effects on HIF-1α siRNA knockdown HCC cells. In addition, genistein enhanced the antitumour effect of sorafenib in sorafenib-resistant HCC cells and HCC-bearing mice.Conclusions:Genistein sensitised aerobic glycolytic HCC cells to apoptosis by directly downregulating HIF-1α, therefore inactivating GLUT1 and HK2 to suppress aerobic glycolysis. The inhibitory effect of genistein on tumour cell growth and glycolysis may help identify effective treatments for HCC patients at advanced stages.

Genistein suppresses the proliferation of telomerase-negative cells.

In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. In this study, we tested genistein, a potential TOP2 inhibitor required for telomere–telomere recombination, on the repression of telomere–telomere recombination. Genistein on the repression of type II recombination on a tlc1 yeast strain was examined by the telomeric DNA structures using Southern blot analysis. Telomere patterns of freshly dissected tlc1 spores containing an empty plasmid (pYES2) or a yeast TOP2 (yTOP2) plasmid were analyzed. The results indicated that the reintroduction of TOP2 recovered the type II pattern, implying genistein in the blockage of type II survivors in the tlc1 strain. The effects of genistein on both tlc1 and tlc1 rad 51 strains in liquid and solid mediums were also examined. Finally, treatment of 10 μmol/L of genistein showed inhibitory effect on the growth of telomerase‐negative U2OS alternative lengthening of telomere (ALT) cells, but not in telomerase‐positive HCT116 cells. These results provide evidences that the inhibitory effects of genistein on telomerase‐negative cells depend on type II recombination pathway in yeast and the ALT pathway in human tumors.

The effect of geniste on Aβ25-35-induced PC12 cell apoptosis through the JNK-dependent Fas pathway.

The β-amyloid protein (Aβ) is considered to be the key factor for inducing Alzheimer's disease (AD). In recent years, the neuroprotective effects of genistein have drawn increasing attention. However, the molecular mechanisms of GEN (genistein) against Aβ are unclear. In the present study, we investigated the inhibitory effects of GEN on Aβ25-35-induced apoptosis in cultured PC12 cells and the related signaling pathway. Our data show that GEN significantly inhibited Aβ25-35-induced apoptosis of PC12 cells. GEN suppressed Aβ25-35-induced JNK activation and the JNK-dependent upregulation of Fas/FasL at the mRNA and protein levels induced by Aβ25-35 were significantly decreased by GEN. Additionally, GEN inhibited mRNA expression and activity of caspase-3 and caspase-8 induced by Aβ25-35. Together, these findings showed that Aβ-induced apoptosis of PC12 cells proceeds through the Fas/FasL pathway. The JNK signaling plays a critical role in regulating the anti-apoptotic effects of genistein. Thus, our results suggest that genistein can inhibit Aβ-induced apoptosis of PC12 cells through blockage of the JNK activation and subsequent suppression of the JNK-dependent Fas/FasL pathway.