Differences in xanthotoxin metabolites in seven mammalian liver microsomes.

Xanthotoxin, abundantly occurring in fruits, vegetables, grapefruit juice and oils, is widely used in medicine for the treatment of psoriasis and vitiligo. Xanthotoxin possesses the ability to inhibit mechanism-based cytochrome P450 (CYP450)-mediated activities in rats and mice. Furthermore, it time-dependently obstructs a number of CYP450-mediated functions in humans. CYP450 enzymes are most abundant in the liver and induce metabolic activation of numerous xenobiotic compounds. The present study aimed to identify the similarities and differences in xanthotoxin metabolism in liver microsomes of 7 mammalian species, including human liver microsomes (HLM), Rhesus monkey liver microsomes (RMLM), Cynomolgus monkey liver microsomes (CMLM), Sprague Dawley rat liver microsomes (RLM), mouse liver microsomes (MLM), Dunkin Hartley guinea pig liver microsomes (PLM) and Beagle dog liver microsomes (DLM). Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometric analysis was used to determine the metabolites. A total of 3 metabolites were detected in RMLM, CMLM and RLM. Furthermore, two metabolites were observed in MLM, HLM, PLM and DLM. By analyzing the type and quantity of metabolites, the metabolism of xanthotoxin in MLM was indicated to be most similar to that in HLM. The metabolic transformations of xanthotoxin in the liver microsomes of the 7 species were analyzed in further detail. On the whole, the results of the present study provide a deeper understanding of the metabolic patterns of xanthotoxin in liver microsomes of different species, which may prove to be advantageous regarding the metabolic mechanisms of action of xanthotoxin. Further insight into drug metabolism with respect to different species will also aid in the selection of appropriate animal models for further research.

Ursolic acid isolated from Isodon excisoides induces apoptosis and inhibits invasion of GBC-SD gallbladder carcinoma cells.

Gallbladder carcinoma (GBC) is a relatively rare but terminal malignancy, and drug/chemical development is an important aspect of prevention and treatment of GBC. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit various pharmaceutical effects. In the present study, the antiproliferative and anti-invasive effects of UA and the associated mechanisms in GBC were examined. UA was isolated from Isodon excisoides. The GBC cells (GBC-SD and NOZ) were treated with UA and subjected to a Cell Counting Kit-8 assay. The GBC-SD cells were subsequently selected for an Annexin V-FITC/propidium iodide assay, Transwell chamber assay, RT2 profiler polymerase chain reaction (PCR) array and western blot analysis. The results indicated that UA inhibited the proliferation and invasion and induced the apoptosis of GBC-SD cells in a dose-dependent manner. Furthermore, the PCR arrays demonstrated that there were 24 differentially expressed genes between the UA-treated and untreated groups. These differentially expressed genes suggested that UA induced the apoptosis of GBC-SD cells through activation of the cell extrinsic pathway. According to Kyoto Encyclopedia of Genes and Genomes pathway analysis of these differentially expressed genes, the suppression of nuclear factor (NF)-κB and protein kinase B (Akt) signaling pathways was further validated. In summary, UA induces the apoptosis and inhibits the invasion of GBC-SD cells, which may be associated with the suppression of NF-κB and Akt signaling pathways. These results may offer a potential therapeutic strategy for the chemoprevention or chemotherapy of GBC in humans.

Effect of ultrasound-assisted extraction on physicochemical properties and TLR2-affinity binding of the polysaccharides from Pholiota nameko.

The effect of the ultrasonic treatment on the physicochemical properties, oil-holding capacities, foaming capacities, and TLR2-affinity of the polysaccharides from Pholiota nameko (PNPS), was evaluated. Compared with the protein content of PNPS before ultrasonic treatment, the protein content of PNPS all presented a decrease under different ultrasonic conditions. The viscosity of PNPS showed a decrease when the ultrasonic intensity was strong enough, as well as the molecular weight. The oil-holding capacity and the foaming capacity of PNPS showed a continuous increasing trend with the increase of the ultrasonic treatment time under a set ultrasonic power of 400 W. Further, the ultrasonic operation could induce the decrease of the affinity binding between PNPS and the receptor proteins TLR2. These data confirmed that applying the ultrasonic treatment could obtain PNPS with high carbohydrate contents, low viscosity and low TLR2-affinity under proper ultrasonic condition.

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole.

1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects. 2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole. 3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1'-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2. 4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.

Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling.

Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-X L , and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis.

Effects of Pholiota nameko polysaccharide on NF-κB pathway of murine bone marrow-derived dendritic cells.

This study investigated the effect of a polysaccharide purified from Pholiota nameko (PNPS-1) on the NF-κB signaling pathway of murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The results showed that PNPS-1 could decrease the expression of maturation markers CD40 and CD80 on BMDCs. PNPS-1 also could decrease the mRNA expression of Myd88, TRAF6, TIRAP, IRAKI, IKBKB, NFKB1, NFKB2 and RelA in immature BMDCs determined by RT-PCR, and decreased the IKKß and P65 production in BMDCs determined by Western blot, and decreased the NF-кB P65 production determined by ELISA. In addition, the effects of PNPS-1 on BMDCs were significantly impaired by treating the cells with anti-TLR2 antibody prior to PNPS-1 treatment, implying direct interaction between PNPS-1 and TLR2 on cell surface. These results indicate that PNPS-1 regulates BMDCs through TLR2 and downstream NF-кB signalings.

Recognization of receptors on bone marrow-derived dendritic cells bound with Pholiota nameko polysaccharides.

Three major active polysaccharides isolated from Pholiota nameko (PNPS), including PNPS-1, PNPS-2 and PNPS-3, had been proved to inhibit the maturation of the murine bone marrow-derived dendritic cells (BMDCs). This paper recognized the affinity bind between PNPS and the five receptors (TLR2, TLR4, CD14, Dectin-1 and Mannose receptor) on BMDCs, using the bio-layer interferometry (BLI)-based biosensor technology developed by ForteBio on Octet RED system (Fortebio, Inc.). From the primary binding experiment, the gradient binding experiment and the inhibition binding experiment between the receptor proteins and PNPS, combined with the binding experiment between PNPS and the BMDCs membranes, we found that PNPS-1, PNPS-2 and PNPS-3 presented strong affinity bind with both TLR2 and Dectin-1 on BMDCs, only PNPS-3 with Mannose receptor. These data confirmed that PNPS could interact with TLR2, Dectin-1 and Mannose receptor that were very important for the affinity bind of these receptors and PNPS, which triggered the further stimulation on BMDCs.

Anticancer activities of citrus peel polymethoxyflavones related to angiogenesis and others.

Citrus is a kind of common fruit and contains multiple beneficial nutrients for human beings. Flavonoids, as a class of plant secondary metabolites, exist in citrus fruits abundantly. Due to their broad range of pharmacological properties, citrus flavonoids have gained increased attention. Accumulative in vitro and in vivo studies indicate protective effects of polymethoxyflavones (PMFs) against the occurrence of cancer. PMFs inhibit carcinogenesis by mechanisms like blocking the metastasis cascade, inhibition of cancer cell mobility in circulatory systems, proapoptosis, and antiangiogenesis. This review systematically summarized anticarcinogenic effect of citrus flavonoids in cancer therapy, together with the underlying important molecular mechanisms, in purpose of further exploring more effective use of citrus peel flavonoids.

Pro-apoptotic effects of the novel tangeretin derivate 5-acetyl-6,7,8,4'-tetramethylnortangeretin on MCF-7 breast cancer cells.

Citrus polymethoxyflavone tangeretin (5,6,7,8,4'-pentamethoxyflavone, TAN) displays multiple biological activities, but previous reports showed that TAN failed to induce MCF-7 human breast cancer cells apoptosis. Herein, we prepared 5-acetyl-6,7,8,4'-tetramethylnortangeretin (5-ATAN), and evaluated its cytotoxicity on MCF-7 cells. 5-ATAN revealed stronger cytotoxicity than that of parent TAN in the growth inhibition of MCF-7 cells. 5-ATAN induced apoptosis via both caspase-independent and -dependent pathways, in which 5-ATAN induced the translocation of apoptosis inducing factor and phosphorylation of H2AX as well as poly (ADP-ribose) polymerase cleavage, caspase-3 activation. However, 5-ATAN did not affect extrinsic markers caspase-8, BID, and FADD. Further, 5-ATAN induced the loss of mitochondrial membrane potential (Δψm) by regulating the Bax/Bcl-2 ratio. Loss of Δψm led to the mitochondrial release of cytochrome c which triggered activation of caspase-9. In conclusion, these data indicate that 5-ATAN plays pro-apoptotic cytotoxic roles in MCF-7 cells through both caspase-dependent intrinsic apoptosis and caspase-independent apoptosis pathways.

Effects of polysaccharides from Pholiota nameko on maturation of murine bone marrow-derived dendritic cells.

This paper studied some structure characters of the Pholiota nameko polysaccharides (PNPS-1), including morphology under SEM and AFM, also the effects of PNPS-1 on the maturation of bone marrow dendritic cells (BMDCs) via concrete changes both inside and outside BMDCs. These impacts on BMDCs were assessed with use of inverted phase contrast microscope for morphology, flow cytometry for key surface molecules, mixed lymphocyte reaction (MLR) for allogeneic T cells proliferation, and bio-assay and enzyme linked immunosorbent assay (ELISA) for cytokine production. We found that PNPS-1 could inhibit phenotypic maturation as evidenced by decreasing expression of CD11c, CD40, CD80, CD83, CD86, and I-A/I-E. Functional maturation inhibition was further confirmed by decreased naive T cell stimulatory activity of BMDCs. Finally, PNPS-1 also stimulated production of more cytokine IL-10 and less IL-12 and TNF-α. These data indicated that PNPS-1 could markedly inhibit the maturation of BMDCs and had potential significant down-regulation immunity.

Effects of the polysaccharide from Pholiota nameko on human cytokine network in serum.

Some physico-chemical characterizations of Pholiota nameko polysaccharides (PNPS-1) were studied, including sulfate content, UV/visible and infrared spectra, also the variation of cytokine communication network in serum to clarify the pharmacological effects of PNPS-1 by determination of 39 cytokines in serum of healthy volunteers. The result proved that PNPS-1 possessed significant anti-inflammatory activity. Further, we use Microsoft Visio 2007 software to map out the cell-cell communication network diagram. The analysis to the diagram suggested that PNPS-1 could take effect on the innate and adaptive immunity and hematopoiesis of volunteers.