Diprenylated flavonoids from licorice induce death of SW480 colorectal cancer cells by promoting autophagy: Activities of lupalbigenin and 6,8-diprenylgenistein.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a well-known herbal medicine, and we previously found that several licorice prenylated flavonoids could cause death of SW480 colorectal cancer cells by promoting autophagy. Given many kinds of prenylated flavonoids in licorice, the activities of other compounds deserve further investigation. In addition, the contribution of isoprenyl groups on the autophagy promotion activities has not been clarified. AIM OF THE STUDY: This study aimed to investigate whether lupalbigenin (LPB) and 6,8-diprenylgenistein (DPG), two licorice diprenylated flavonoids, could induce autophagic cell death of SW480 cells, and clarify the contribution of isoprenyl groups. MATERIALS AND METHODS: Cytotoxic activities of LPB and DPG were tested by using an MTT method, and apoptosis induction effects were evaluated by PI-Annexin V staining-based flow cytometry and protein levels of caspase-3 and PARP-1. Autophagy promotion effects of LPB and DPG were assessed by protein levels of LC3, p62, Akt and mTOR as well as number of autophagosomes in cells, and autophagy inhibitor chloroquine (CQ) was involved to identify the role of autophagy on LPB or DPG-caused death of SW480 cells. In addition, two groups of structurally similar diprenylated, mono-prenylated and free flavonoids were obtained from licorice, which were used to investigate the contribution of isoprenyl groups on their autophagy promotion activities. RESULTS: Both LPB and DPG significantly induced apoptosis of SW480 cells with strong cytotoxic activities, and meanwhile, they also promoted autophagy probably through the Akt/mTOR signaling pathway. Further studies indicated that LPB and DPG could induce autophagic cell death of SW480 cells. Moreover, isoprenyl groups contributed mainly to the cytotoxic and autophagy promotion activities of licorice prenylated flavonoids. CONCLUSION: This study reported for the first time that licorice diprenylated flavonoids LPB and DPG induced death of SW480 cells by promoting autophagy, which was mainly attributed to the isoprenyl groups. The results provided theoretical basis for researches on anti-colorectal cancer drugs and their structural modification.

Genistein: A Potent Anti-Breast Cancer Agent.

Genistein is an isoflavonoid present in high quantities in soybeans. Possessing a wide range of bioactives, it is being studied extensively for its tumoricidal effects. Investigations into mechanisms of the anti-cancer activity have revealed many pathways including induction of cell proliferation, suppression of tyrosine kinases, regulation of Hedgehog-Gli1 signaling, modulation of epigenetic activities, seizing of cell cycle and Akt and MEK signaling pathways, among others via which the cancer cell proliferation can be controlled. Notwithstanding, the observed activities have been time- and dose-dependent. In addition, genistein has also shown varying results in women depending on the physiological parameters, such as the early or post-menopausal states.

7-Difluoromethoxy-5,4'-dimethoxy-genistein attenuates macrophages apoptosis to promote plaque stability via TIPE2/TLR4 axis in high fat diet-fed ApoE-/- mice.

Promoting plaque stability is of great significance for prevention and treatment of cardiovascular diseases. 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) is a novel active compound synthesized using genistein, which exerts anti-atherosclerotic effect. In this study, we evaluated effects of DFMG on plaque stability in ApoE-/- mice fed with high fat diet (HFD), and explored the molecular mechanism by using ApoE-/-TLR4-/- mice and RAW264.7 cells. Here, we found that DFMG significantly reduced plaque areas, macrophages infiltration and apoptosis, and TLR4 expression in HFD-fed ApoE-/- mice. Meanwhile, DFMG increased collagen fibers, smooth muscle cells and TIPE2 expression in plaques and media. Besides, TLR4 knockout promoted the protective effects of DFMG on plaques. In vitro, DFMG decreased lysophosphatidylcholine (LPC)-induced macrophages apoptosis and TLR4, while upregulated TIPE2. Moreover, TIPE2 reduced TLR4, MyD88, p-NF-κB p65Ser276, cleaved Caspase-3 overproduction, and enhanced effects of DFMG on LPC-induced macrophages. Overall, our study demonstrates that DFMG can promote plaque stability by reducing macrophage apoptosis through TIPE2/TLR4 signaling pathway, which suggests DFMG should be used to develop food additives or drugs for preventing atherosclerosis.

Genistein-3'-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway.

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3'-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1ß level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.

Effects of 6,8-Diprenylgenistein on VEGF-A-Induced Lymphangiogenesis and Lymph Node Metastasis in an Oral Cancer Sentinel Lymph Node Animal Model.

BACKGROUND: The major determining factor of prognosis of oral squamous cell carcinoma is cervical lymph node metastasis. 6,8-Diprenylgenistein (6,8-DG), an isoflavonoid isolated from Cudrania tricuspidata has been reported to have anti-microbial and anti-obesity activities. However, its effects on lymphangiogenesis and lymph node metastasis in oral cancer have not yet been reported. METHODS: To investigate the in vitro inhibitory effects of 6,8-DG on VEGF-A-induced lymphangiogenesis, we performed the proliferation, tube formation, and migration assay using human lymphatic microvascular endothelial cells (HLMECs). RT-PCR, Western blot, immunoprecipitation, ELISA and co-immunoprecipitation assays were used to investigate the expression levels of proteins, and mechanism of 6,8-DG. The in vivo inhibitory effects of 6,8-DG were investigated using an oral cancer sentinel lymph node (OCSLN) animal model. RESULTS: 6,8-DG inhibited the proliferation, migration and tube formation of rhVEGF-A treated HLMECs. In addition, the in vivo lymphatic vessel formation stimulated by rhVEGF-A was significantly reduced by 6,8-DG. 6,8-DG inhibited the expression of VEGF-A rather than other lymphangiogenic factors in CoCl2-treated SCCVII cells. 6,8-DG inhibited the expression and activation of VEGFR-2 stimulated by rhVEGF-A in HLMECs. Also, 6,8-DG inhibited the activation of the lymphangiogenesis-related downstream signaling factors such as FAK, PI3K, AKT, p38, and ERK in rhVEGF-A-treated HLMECs. Additionally, 6,8-DG inhibited the expression of the hypoxia-inducible factor (HIF-1α), which is involved in the expression of VEGF-A in CoCl2-treated SCCVII cells, and 6,8-DG inhibited VEGF-A signaling via interruption of the binding of VEGF-A and VEGFR-2 in HLMECs. In the VEGF-A-induced OCSLN animal model, we confirmed that 6,8-DG suppressed tumor-induced lymphangiogenesis and SLN metastasis. CONCLUSION: These data suggest that 6,8-DG inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in vitro and in vivo. Furthermore, the inhibitory effects of 6,8-DG are probably mediated by inhibition of VEGF-A expression in cancer cells and suppression of the VEGF-A/VEGFR-2 signaling pathway in HLMEC. Thus, 6,8-DG could be novel and valuable therapeutic agents for metastasis prevention and treatment of oral cancer.

8-prenylgenistein exerts osteogenic effects via ER α and Wnt-dependent signaling pathway.

8-prenylgenistein (8PG) was previously reported to exert stronger osteogenic activity than genistein, a well-known soy phytoestrogen. However, the molecular mechanism underlying the actions of 8PG on osteoblasts was far from clear. In the present study, the osteogenic effects and mechanisms of 8PG and genistein were studied using human BMSC and murine pre-osteoblast MC3T3-E1 cells. Our results indicated that the stimulatory effects of 8PG and genistein on osteoblast differentiation were abolished by co-incubation with MPP (10-6 M, an ERα antagonist), but not PHTPP (10-6 M, an ERß antagonist). Molecular docking indicated that the binding mode of 8PG toward ERα was similar to that of genistein and therefore could not account for their differential osteogenic actions. In silico target profiling identified the involvement of glycogen synthase kinase-3ß (GSK-3ß), a key mediator of Wnt/ß-catenin pathway, in the actions of 8PG. However, instead of directly inhibiting GSK-3ß enzymatic activities, 8PG and genistein were found to induce GSK-3ß phosphorylation at Serine-9 in osteoblastic MC3T3-E1 cells. 8PG exerted more potent effects than genistein in stimulating expressions of LRP5, ß-catenin, Runx2, osteocalcin, alp, opg, major protein and gene markers involved in Wnt signaling pathway in MC3T3-E1 cells. Moreover, the inhibition of Wnt signaling by DKK1 could be restored by treatment with 8PG and genistein. However, 8PG, but not genistein, stimulated ERα-dependent ß-catenin protein expression in MC3T3-E1 cells. Furthermore, the increase in ALP activity, LRP5 and phospho-Akt/Akt expression by 8PG and genistein were abolished by co-treatment with LY294002 (10-5 M, a PI3K pathway inhibitor). Collectively, our results suggested that the osteogenic activities of 8PG was mediated by GSK-3ß phosphorylation through the induction of Wnt/ß-catenin and ERα-associated PI3K/Akt signaling.

Structure-bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell.

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram'-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.

7­Difluoromethyl­5,4'­dimethoxygenistein exerts anti­angiogenic effects on acute promyelocytic leukemia HL­60 cells by inhibiting the TLR4/NF­κB signaling pathway.

Angiogenesis plays an important role in the development and metastasis of tumors, and anti-angiogenesis agents are used to treat tumors. For example, the acute promyelocytic leukemia (APL) may be treated with arsenic trioxide. Angiogenesis in APL is a multi­step dynamic equilibrium process coordinated by various angiogenic stimulators and inhibitors, which play key roles in the occurrence, progression and chemosensitivity of this disease. Our research group previously synthesized 7­difluoromethyl­5,4'­dimethoxygenistein (DFMG), and found that it inhibits angiogenesis during atherosclerotic plaque formation. In the present study, the effect and mechanism of DFMG in angiogenesis induced by APL HL­60 cells was investigated using a chick embryo chorioallantoic membrane model and Matrigel tubule formation assays. The results obtained revealed an anti­angiogenesis effect of DFMG towards HL­60 cells. When the Toll­like receptor 4/nuclear factor­κB (TLR4/NF­κB) signaling pathway was inhibited, the anti­angiogenic effect of DFMG was further enhanced. However, when the TLR4/NF­κB signaling pathway was activated, the anti­angiogenic effect of DFMG was attenuated. These results demonstrated that DFMG inhibits angiogenesis induced by APL HL­60 cells, and provides insights into the mechanism by which DFMG inhibits the TLR4/NF­κB signaling pathway. In conclusion, in the present study, the anti­angiogenesis effect of DFMG on APL has been reported, and the mechanism by which DFMG induced the anti­angiogenesis effect was explored. These findings have provided a potential new drug candidate for the treatment of patients with APL.

Disruption of crosstalk between LX-2 and liver cancer stem-like cells from MHCC97H cells by DFOG via inhibiting FOXM1.

Hepatic stellate cell (HSC) line LX-2 is activated by liver cancer stem-like cells (LCSLCs) and produces various cytokines that make up most of the hepatocellular carcinoma (HCC) microenvironment. The new genistein derivative, 7-difluoromethoxyl-5,4'-di-n-octylgenistein (DFOG), shows anticancer effects in multiple malignancies by controlling forkhead box M1 (FOXM1). In this study, we aimed to assess whether DFOG disrupts the crosstalk between human HSC LX-2 cells and LCSLCs. Distinct generations of MHCC97H-derived spheres were obtained with the second generation considered as LCSLCs which displayed enhanced self-renewal ability and elevated expression levels of CD133, CD44, and EpCAM proteins, as well as tumorigenicity, as revealed by colony formation assay in vitro and tumorigenicity assay in vivo. LX-2 and MHCC97H cells were co-cultured with/without DFOG (1, 5, and 10 µM, respectively) using the transwell system. FOXM1 overexpression and/or knockdown were employed for mechanistic investigations. Our results suggested that Co-CM promoted LX-2 cell transformation into liver cancer-associated HSCs. Meanwhile, FOXM1 was up-regulated and the level of hepatocyte growth factor (HGF) was increased in LX-2 cells and in the supernatant after Co-CM stimulation. Sphere and colony formation abilities in MHCC97H cells, and protein levels of CD133, CD44, and EpCAM, were also markedly elevated. DFOG dose-dependently inhibited the above effects, similar to FOXM1 knockdown in LX-2 cells. FOXM1 overexpression reversed the inhibitory effects of DFOG or FOXM1 knockdown or both on LX-2 cell activation and LCSLC feature induction in MHCC97H cells by LCSLC/LX-2 co-culture. This study demonstrated that DFOG disrupts the crosstalk between HSCs and LCSLCs to suppress LCSLC features via down-regulating FOXM1 expression and reducing HGF secretion in HSCs.

Clinical candidate and genistein analogue AXP107-11 has chemoenhancing functions in pancreatic adenocarcinoma through G protein-coupled estrogen receptor signaling.

Despite advances in cancer therapeutics, pancreatic cancer remains difficult to treat and often develops resistance to chemotherapies. We have evaluated a bioavailable genistein analogue, AXP107-11 which has completed phase Ib clinical trial, as an approach to sensitize tumor cells to chemotherapy. Using organotypic cultures of 14 patient-derived xenografts (PDX) of pancreatic ductal adenocarcinoma, we found that addition of AXP107-11 indeed sensitized 57% of cases to gemcitabine treatment. Results were validated using PDX models in vivo. Further, RNA-Seq from responsive and unresponsive tumors proposed a 41-gene treatment-predictive signature. Functional and molecular assays were performed in cell lines and demonstrated that the effect was synergistic. Transcriptome analysis indicated activation of G-protein-coupled estrogen receptor (GPER1) as the main underlying mechanism of action, which was corroborated using GPER1-selective agonists and antagonists. GPER1 expression in pancreatic tumors was indicative of survival, and our study proposes that activation of GPER1 may constitute a new avenue for pancreatic cancer therapeutics.

Development of monoclonal antibody-based enzyme-linked immunosorbent assay for quantitative quality control of Derris scandens (Roxb.) Benth.

Derris scandens (Roxb.) Benth. is a medicinal plant used for treatment of musculoskeletal pain in Thai traditional medicines. Its stem contains active compound genistein-7-O-[α-rhamnopyranosyl-(1 to 6)-ß-glucopyranoside] (GTG) which is used as a biomarker for standardization of D. scandens extracts. As an alternative for rapid quantitation of GTG, a monoclonal antibody against GTG was prepared and applied for an indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine GTG in plants and herbal products. The established method provided a quantification range of 0.31-10 µg/mL with a limit of detection of 0.29 µg/mL. The assay was validated for precision and accuracy by intra- and interassay variation analyses, recovery test, and comparison analysis between the amounts of GTG determined by ELISA and HPLC. The results exhibited that the developed ELISA is sensitive and effective for determination of GTG in D. scandens plant materials and herbal products.

Differential effects of genistein and 8-prenylgenistein on reproductive tissues in immature female mice.

CONTEXT: We identified an active prenylated derivative of genistein, 8-prenylgenistein (8PG) from Erythrina variegata L. (Leguminosae) and found that 8PG increased osteoprotective effects of genistein in oestrogen-deficient mice. OBJECTIVE: This study investigated and compared the oestrogenic effects of genistein and 8PG on uterus and vagina of immature mice. MATERIALS AND METHODS: Immature female CD-1 mice were orally treated with vehicle (Control, n = 10) or genistein (75 mg/kg, n = 10) or 8PG with low (8PG-L, 75 mg/kg, n = 10) and high dose (8PG-H, 150 mg/kg, n = 10) for 7 consecutive days by intragastric gavage. The uterus and vagina were harvested for histological and molecular measurements. RESULTS: Treatment with genistein and 8PG-H significantly increased uterus index (1.98 ± 0.21 & 1.49 ± 0.16 mg/g) and vagina index (3.83 ± 0.11 & 3.13 ± 0.25 mg/g) as compared to untreated control (uterus, 1.12 ± 0.13 mg/g; vagina, 2.32 ± 0.18 mg/g). Accordingly, both genistein and 8PG-H made vaginal cells keratinized and induced uterine and vaginal hypertrophy associated with the endometrial proliferation. 8PG-L did not affect oestrus cycle and histology of uterus and vagina. Treatment of immature mice with genistein or 8PG-H upregulated protein expression of oestrogen receptor-α (ER-α) and proliferating cell nuclear antigen (PCNA), but 8PG-L did not alter ER-α and PCNA expression in uterus and vagina. CONCLUSION: This study indicated that 8-prenylgenistein exerted oestrogenic effects in immature female mice. The efficacy and safety of 8-prenylgenistein when applied in improving oestrogen deficiency-induced syndrome requires further elucidation.

GEN-27 exhibits anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway.

Prolonged inflammation and deregulated cytokine production are associated with diversified inflammatory diseases. Genistein (GEN), the active and predominant isoflavonoid in dietary soybean, possesses anti-inflammatory activity. Our study aimed to assess the anti-inflammatory effects of GEN-27, a derivative of GEN, as well as explore the potential molecular mechanisms using lipopolysaccharide (LPS)-induced RAW264.7 cells. In our study, we demonstrated that GEN-27 administration (1, 5, or 10 µM) dose-dependently inhibited nitrite and nitric oxide (NO) levels in LPS-stimulated RAW264.7 cells. Also, GEN-27 suppressed the release of LPS-induced pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and IL-18. Moreover, GEN-27 attenuated LPS-induced inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) expressions at messenger RNA and protein levels, and reversed the promoter activity of iNOS in RAW264.7 cells. Mechanistically, GEN-27 abated LPS-induced reactive oxygen species production, as well as mitigated LPS-induced increase of caspase 1 activity and the protein levels of NOD-like receptor 3 (NLRP3), anti-apoptosis-associated speck-like protein-containing a CRAD (ASC), and caspase 1 in RAW264.7 cells in a dose-dependent manner. Similarly, GEN-27 dose-dependently weakened adenosine triphosphate-induced NLRP3 and IL-1ß in RAW264.7 cells. In addition, GEN-27 treatment significantly suppressed LPS-induced phosphorylation of nuclear factor-κB (NF-κB) p65 and alleviated LPS-induced increase of transcriptional activity of NF-κB in RAW264.7 cells. In summary, these results revealed that GEN-27 exhibited anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway, suggesting that GEN-27 may be served as a promising therapeutic agent for the prevention and therapy of inflammatory-associated diseases.

5-Hydroxy-4'-Nitro-7-Propionyloxy-Genistein Inhibited Invasion and Metastasis via Inactivating Wnt/b-Catenin Signal Pathway in Human Endometrial Carcinoma Ji Endometrial Cells.

BACKGROUND Chemotherapy has been assuring more important roles in the treatment of carcinoma. Developing new types of drugs with less adverse effects and low drug resistance has become an important researching focus. The present study aimed to investigate the anticancer effects of 5-hydroxy-4'-nitro-7-propionyloxy-genistein (HNPG) and to elucidate its underlying molecular mechanism. MATERIAL AND METHODS The inhibitory effects of cell viability of HNPG were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flat plate clone formation method, and Transwell assay. The distribution of cell cycle was analyzed using flow cytometry (FCM) method. The morphological alteration, root-mean-squared roughness (Rq), average roughness (Ra), Young's modulus, and adhesive force were measured by atomic force microscope (AFM) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were used to explore the possible molecular mechanism. RESULTS We found that HNPG had dramatic activity against Ji Endometrial cells (JEC) in vitro, inhibited the proliferation and colony formation, attenuated invasion and migration ability, and arrested cell cycle in G1 phase, all in a dose-dependent manner. Simultaneously, cell bodies shrunk, pseudopod structures retracted, Rq and Ra were reduced, and Young's modulus and adhesive force increased, accompanied by downregulation of ß-catenin, C-Myc, Cyclin D1, matrix metalloprotease 2 (MMP-2), matrix metalloprotease 7 (MMP-7), and matrix metalloprotease 9 (MMP-9). CONCLUSIONS HNPG dramatically inhibited invasion and metastasis of JEC cells in vitro. Its molecular mechanism might be related to inactivation of the wnt/ß-catenin signal pathway, accumulated cells in G1/S phase, inhibited cell proliferation, improved adhesive force between cells, and reduced cell plasticity and elasticity.

[Effects of 7-difluoromethy-5, 4'-dimethoxygenistein on stress urinary incontinence model in rats and its mechanisms].

OBJECTIVE: To investigate the effects of 7-difluoromethy-5, 4'-dimethoxygenistein (DFMG) on stress urinary incontinence (SUI) model in Sprague Dawley (SD) rats and its possible mechanisms.
 Methods: SD rat model of SUI was established through simulating pregnancy, birth trauma and ovarian castration. The rats were divided into a normal control group, a SUI group, and a DFMG group at 10 or 20 mg/kg. They were treated with 10 mg/kg normal saline (NS), 10 mg/kg NS, 10 mg/kg DFMG and 20 mg/kg DFMG, respectively, via gastric gavage every other day. Maximal bladder capacity (MBC), leak point pressure (LPP), abdominal leak point pressure (ALPP), hematoxylin-eosin (HE) staining, and Masson staining were performed to detect the index for the model. MiR-26b and its down-stream gene phosphatase and tensin homolog deleted on chromosome 10 (PENT) mRNA in urethral sphincter muscles cells (USMCs) were analyzed by RT-PCR. The protein levels of PENT, phosphatidylinositol 3-kinase (PI3K), protein kinaseB (AKT), B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome C(Cyt-c) and caspase-3 were examined by Western blot. The apoptotic rate of USMCs was determined by flow cytometry (FCM), and the proliferative rate of USMCs was examined by MTT assay.
 Results: The SD rat model of SUI was successfully established. HE staining and Masson staining showed that the pathological features of urethral sphincter were improved in the DFMG-treated groups compared with the SUI group. The urine dynamics indexes of model rats, such as MBC, LPP and ALPP, were improved (all P﹤0.05). The results of RT-PCR showed that the miR-26b mRNA was up-regulated (P﹤0.05) and PENT mRNA was down-regulated (P﹤0.05) in the DFMG-treated groups compared with the SUI group. Simultaneously, compared with the SUI group, the protein levels of PENT, Bax, Cyt-c and caspase-3 were down-regulated (all P﹤0.05) and the protein levels of PI3K, AKT and Bcl-2 protein were up-regulated (all P﹤0.05), accompanied by the decreased apoptotic rate of USMCs (P﹤0.05) and the increased proliferative rate of USMCs (P﹤0.05) in the DFMG-treated groups.
 Conclusion: The DFMG can significantly improve the symptoms of urinary dynamics, which might be related to the up-regulation of miR-26b expression and the regulation of PI3/AKT-Bcl-2/ Bax signaling pathways.

DFMG attenuates the activation of macrophages induced by co­culture with LPC­injured HUVE­12 cells via the TLR4/MyD88/NF­κB signaling pathway.

7­difluoromethoxy­5,4'­dimethoxy­genistein (DFMG) is a novel active chemical entity, which modulates the function and signal transduction of endothelial cells and macrophages (MPs), and is essential in the prevention of atherosclerosis. In the present study, the activity and molecular mechanism of DFMG on MPs was investigated using a Transwell assay to construct a non­contact co­culture model. Human umbilical vein endothelial cells (HUVE­12), which were incubated with lysophosphatidylcholine (LPC), were seeded in the upper chambers, whereas PMA­induced MPs were grown in the lower chambers. The generation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) were measured using the corresponding assay kits. The proliferation and migration were assessed using 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide and wound healing assays, respectively. Foam cell formation was examined using oil red O staining and a total cholesterol assay. The protein expression levels of Toll­like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)­κB p65 were detected by western immunoblotting. The secretion of interleukin (IL)­1ß was examined using an enzyme­linked immunosorbent assay. It was found that LPC significantly increased the generation of ROS and the release of LDH in HUVE­12 cells. The LPC­injured HUVE­12 cells activated MPs under co­culture conditions and this process was inhibited by DFMG treatment. LPC upregulated the expression levels of TLR4, MyD88 and NF­κB p65, and the secretion of IL­1ß in the supernatant of the co­cultured HUVE­12 cells and MPs. These effects were reversed by the application of DFMG. Furthermore, CLI­095 and IL­1Ra suppressed the activation of MPs that was induced by co­culture with injured HUVE­12 cells. These effects were further enhanced by co­treatment with DFMG, and DFMG exhibited synergistic effects with a TLR4­specific inhibitor. Take together, these findings revealed that DFMG attenuated the activation of MP induced by co­culture with LPC­injured HUVE­12 cells. This process was mediated via inhibition of the TLR4/MyD88/NF­κB signaling pathway in HUVE­12 cells.

DFMG reverses proliferation and migration of vascular smooth muscle cells induced by co-culture with injured vascular endothelial cells via suppression of the TLR4-mediated signaling pathway.

7-Difluoromethoxy-5,4'-dimethoxy-genistein (DFMG) is a novel chemical compound synthesized using genistein. Previous studies have indicated that DFMG can reverse the apoptosis of vascular endothelial cells (VECs) by regulating the mitochondrial apoptosis pathway. The present study aimed to investigate the activity and molecular mechanism underlying DFMG­mediated protection of vascular smooth muscle cell (VSMCs) using a non­contact co­culture model established by using Transwell insert. Secretion of interleukin­6 (IL­6) and tumor necrosis factor­α (TNF­α) were measured by ELISA. Proliferation and migration of VSMCs were assessed using a Cell Counting kit­8 and wound healing assays, respectively. Toll­like receptor 4 (TLR4) mRNA and protein levels were detected by reverse transcription-quantitative polymerase chain reaction and western blotting analyses, respectively. In the present study, lysophosphatidylcholine (LPC) significantly increased the secretion of IL­6 and TNF­α in VECs. VECs treated with LPC markedly increased proliferation and migration of VSMCs, which were inhibited by DFMG. Transfection of either TLR4 short hairpin RNA (shRNA) or TLR4 cDNA in VECs inhibited and increased proliferation and migration of VSMCs, respectively. Furthermore, transfection of VECs with TLR4 shRNA suppressed the proliferation and migration of VSMCs induced by co­culture with injured VECs, which was further enhanced by treatment with DFMG. By contrast, transfection of VECs with TLR4 cDNA enhanced proliferation and migration of VSMCs and this effect was inhibited by treatment with DFMG. Taken together, the results of the present study demonstrated that DFMG can reverse proliferation and migration of VSMCs induced by co­culture with injured VECs via suppression of the TLR4­mediated signaling pathway.

Targeted Metabolomics Identifies Pharmacodynamic Biomarkers for BIO 300 Mitigation of Radiation-Induced Lung Injury.

PURPOSE: Biomarkers serve a number of purposes during drug development including defining the natural history of injury/disease, serving as a secondary endpoint or trigger for intervention, and/or aiding in the selection of an effective dose in humans. BIO 300 is a patent-protected pharmaceutical formulation of nanoparticles of synthetic genistein being developed by Humanetics Corporation. The primary goal of this metabolomic discovery experiment was to identify biomarkers that correlate with radiation-induced lung injury and BIO 300 efficacy for mitigating tissue damage based upon the primary endpoint of survival. METHODS: High-throughput targeted metabolomics of lung tissue from male C57L/J mice exposed to 12.5 Gy whole thorax lung irradiation, treated daily with 400 mg/kg BIO 300 for either 2 weeks or 6 weeks starting 24 h post radiation exposure, were assayed at 180 d post-radiation to identify potential biomarkers. RESULTS: A panel of lung metabolites that are responsive to radiation and able to distinguish an efficacious treatment schedule of BIO 300 from a non-efficacious treatment schedule in terms of 180 d survival were identified. CONCLUSIONS: These metabolites represent potential biomarkers that could be further validated for use in drug development of BIO 300 and in the translation of dose from animal to human.

Inactivation of AKT, ERK and NF-κB by genistein derivative, 7-difluoromethoxyl-5,4'-di-n-octylygenistein, reduces ovarian carcinoma oncogenicity.

Cancer stem cells (CSCs) have central functions in cancer formation and development. Aberrant expression of AKT, ERK and NF-κB signaling pathways have been reported in several types of CSCs. Phytochemicals from dietary compounds possess anti-CSC properties, and have been characterized as promising therapeutic agents for the prevention and treatment of many types of cancers. We previously showed that the newly synthesized genistein derivative, 7-difluoromethoxyl-5,4'-di-n-octylygenistein (DFOG), can inhibit the self-renewal ability of ovarian cancer stem cells (OVCSLCs). In the present study, we further assessed whether various signaling pathways are regulated by DFOG. We found that spheroids derived from the SKOV3 cell line possessed OVCSLC properties and DFOG efficiently inhibited the stemness of the OVCSLCs. In addition, the suppression of spheroid and colony formation by DFOG was associated with inhibition of AKT and ERK1/2 protein phosphorylation, and NF-κB activity in OVCSLCs from the SKOV3 cells. Importantly, DFOG inhibited the oncogenicity of the OVCSLCs by activation of FoxO3a and/or inactivation of FoxM1 by the targeting of multiple pro-survival (AKT and ERK1/2) and proinflammatory (NF-κB) pathways, providing a new avenue for the treatment of ovarian carcinoma in humans.

Spectroscopic and molecular docking study on the structure-affinity relationship and mechanism in the interaction of genistein and its derivatives with bovine serum albumin.

In this paper, the interaction of genistein (GEN) and its four derivatives (GEN1-4) with bovine serum albumin (BSA) were investigated by ultraviolet-visible absorption spectra, fluorescence, synchronous fluorescence, three-dimensional fluorescence spectroscopy, circular dichroism and molecular docking techniques. The experimental results showed that the intrinsic fluorescence of BSA was quenched by genisteins and was due to the formation of a genisteins-BSA complex. The quenching constant, binding constants, binding sites, intermolecular distances and thermodynamic properties were calculated at 298 K, 306 K and 310 K. Site marker competitive experiments indicated that the binding site of genisteins to BSA was mainly located in subdomain IIA. The conformational investigation showed that the presence of 0020 genisteins led to changes in the secondary structure of BSA and induced the slight unfolding of protein polypeptides, which confirmed some micro-environmental and conformational changes of BSA molecules. Furthermore, the binding affinity decreased in the order GEN1 > GEN > GEN4 > GEN3 > GEN2, which revealed that different type and position of substituents of genistein significantly influenced the affinity of compounds to BSA. The number of hydroxyl groups on the ring A was the most important factor because increasing the hydroxyl groups on ring A clearly enhanced the binding affinity. However, trifluoromethylation did not much affect the affinity, alkylation, esterification and difluoromethylation slightly enhanced the binding affinity. The results obtained herein will provide valuable information about the pharmacokinetics at a molecular level and be a useful guideline for the further design of much more suitable genistein derivatives.