Hesperetin upregulates ABCA1 expression and promotes cholesterol efflux from THP-1 macrophages.

ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR). Since ABCA1 plays a pivotal role in cholesterol and HDL metabolism, identification of a compound capable of increasing its expression may be beneficial for the prevention and therapy of atherosclerosis. Firefly luciferase reporter assays were developed for human ABCA1 promoters and LXR enhancers, and an in-house phytochemical library was screened. It was found that a citrus flavonoid, hesperetin (1), increased ABCA1 promoter and LXR enhancer activities in THP-1 macrophages. It was also found that this flavonoid promoted PPAR-enhancing activity. In accordance with these findings, 1 increased mRNA and protein expression of ABCA1 and consequently upregulated ApoA-I-mediated cholesterol efflux. These results provide evidence that 1 promotes ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression through the activation of LXRα and PPARγ.

Ethanolic extracts of Brazilian red propolis increase ABCA1 expression and promote cholesterol efflux from THP-1 macrophages.

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by regulating the cellular efflux of cholesterol. Since ABCA1 plays a pivotal role in cholesterol homeostasis and HDL metabolism, identification of a novel substance that is capable of increasing its expression would be beneficial for the prevention and therapy of atherosclerosis. In the present study, we studied the effects of ethanolic extracts of Brazilian red propolis (EERP) on ABCA1 expression and cholesterol efflux in THP-1 macrophages. EERP enhanced PPARγ and liver X receptor (LXR) transcriptional activity at 5-15µg/ml, which was associated with upregulation of PPARγ and LXRα expression. It was also found that EERP increase the activity of the ABCA1 promoter, which is positively regulated by LXR. Consistent with these findings, treatment with EERP increased both mRNA and protein expression of ABCA1. Finally, EERP upregulated ApoA-I-mediated cholesterol efflux. Our results showed that EERP promote ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression via induction of PPARγ/LXR.

Isolation of chemical constituents from Enicosanthum cupulare (King) Airy-Shaw.

Six chemical constituents were isolated from Enicosanthum cupulare (King) Airy-Shaw. The structures of the isolated compounds were determined using 1D, 2D-NMR and were found to be identical to those previously reported for these compounds. Three aporphine alkaloids, O-methylmoschatoline, liriodenine and oxostephanine were isolated from this plant for the first time. In addition, we examined the effects of these compounds on melanogenesis in murine B16 melanoma cells; O-methylmoschatoline increased melanin content in these cells.

Resveratrol oligomers from Vatica albiramis.

Five new stilbenoids, vatalbinosides A-E (1-5), and 13 known compounds (6-18) were isolated from the stem of Vatica albiramis. The effects of these new compounds on interleukin-1ß-induced production of matrix metalloproteinase-1 (MMP-1) in human dermal fibroblasts were examined. Three resveratrol tetramers, (-)-hopeaphenol (6), vaticanol C (13), and stenophyllol C (14), were identified as strong inhibitors of MMP-1 production.

Inhibitory effects of whisky congeners on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

Whisky is matured in oak casks. Many nonvolatile substances (whisky congeners, WC) seep from the oak cask during the maturing process. In this study, three antiallergic agents (syringaldehyde, SA; lyoniresinol, Lyo; and ellagic acid, EA) were isolated from WC. Treatment with SA, Lyo, and EA reduced the elevation of intracellular free Ca(2+) concentration ([Ca(2+)]i) and intracellular ROS production caused by FcepsilonRI activation. The inhibitions of the elevation of [Ca(2+)]i and intracellular ROS production by SA and Lyo were mainly due to the suppression of the NADPH oxidase activity and scavenging of the produced radical, respectively. On the other hand, EA inactivated spleen tyrosine kinase and led to the inhibition of the elevation of [Ca(2+)]i and intracellular ROS production. Furthermore, it was found that WC strongly inhibited IgE binding to the FcepsilonRIalpha chain, whereas SA, Lyo, and EA did not indicate this inhibitory effect. These results suggest that WC inhibits allergic reactions through multiple mechanisms. To disclose the in vivo effects of WC, SA, Lyo, and EA, these compounds were administered to type I allergic model mice, and the passive cutaneous anaphylaxis (PCA) reaction was measured. These compounds remarkably suppressed the PCA reaction. Taken together, these findings suggest that WC seemed to be beneficial to ameliorate allergic reactions.

Ethanolic extracts of Brazilian red propolis promote adipocyte differentiation through PPARγ activation.

AIM OF THE STUDY: The aim of present study was to investigate the effects of ethanolic extracts of red propolis (EERP) on adipogenesis and evaluate the molecular basis for their anti-obesity effects. MATERIALS AND METHODS: We tested whether EERP alone could induce differentiation of 3T3-L1 cells, regulate the expression of adipocyte-specific genes and reverse inhibitory effects of TNF-α on their differentiation. Next, we performed a luciferase reporter gene assay to test whether EERP could enhance transcriptional activities of PPARγ and adiponectin promoter activities. RESULTS: EERP strongly induced differentiation of 3T3-L1 preadipocytes into adipocytes, and enhanced the PPARγ transcriptional activity and adiponectin promoter activity. In addition, EERP attenuated the inhibitory effect of TNF-α on adipocyte differentiation and adiponectin production in mature adipocytes. CONCLUSION: The present study indicates that EERP enhance differentiation of 3T3-L1 adipocytes in part by its potency of PPARγ activation and are capable of reversing inhibitory effects of TNF-α on adipocyte differentiation and adiponectin expression. These results suggest the value of EERP as a diet supplement for prevention and treatment of obesity and obesity-associated disorders.

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki 'Fuyu') on melanin biosynthesis.

We found that the acetone extract of the peel of Japanese persimmon (Diospyros kaki 'Fuyu') inhibits melanin biosynthesis in mouse B16 melanoma cells. The activity-guided purification of the extract resulted in isolation of two active compounds, which have been identified as flavonoid glycosides, isoquercitrin (quercetin-3-O-glucoside) and hyperin (quercetin-3-O-galactoside) by spectral analysis. Isoquercitrin and hyperin strongly inhibited the production of melanin (IC(50): 21.7 and 18.2 microM, respectively). The inhibitory effects were found to be mediated by suppression of tyrosinase expression.

Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1.

A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPbeta, PLD1, and C/EBPalpha which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX + any other inducer induced mild adipocyte differentiation, whereas insulin + dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPbeta, but not C/EBPalpha, increased PLD1 mRNA and PLD1 5' promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPbeta, but not C/EBPalpha, to these C/EBP motifs of PLD1 5' promoter. Our results show that PLD1 is a target gene of C/EBPbeta through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

Effects of theaflavins on melanin biosynthesis in mouse b16 melanoma cells.

In this study, we examined the effects of theaflavins, polyphenols in black tea, on alpha melanocyte-stimulating hormone (alphaMSH)-induced melanogenesis in mouse B16 melanoma cells. Among theaflavins, theaflavin-3,3'-digallate (TF-3) exhibited a potent inhibitory effect on melanogenesis. Our results indicate that the inhibitory effect of TF-3 on melanogenesis is due to suppression of tyrosinase protein and mRNA levels.

Intracellular glutathione regulates sesquiterpene lactone-induced conversion of autophagy to apoptosis in human leukemia HL60 cells.

A sesquiterpene lactone, eupalinin A, found in Eupatorium chinense L., exhibited a marked inhibitory effect on cell growth in HL60 cells. In a previous study, it was indicated that the intracellular ROS generation accompanying mitochondrial dysfunction was closely related to the autophagic cell death induced by the treatment with eupalinin A. By glutathione (GSH) pre-treatment, eupalinin A-induced cell growth inhibition was markedly reduced in a time- and dose-dependent manner. Eupalinin A reduced the intracellular GSH level in the early phase, but the GSH pre-treatment reduced this depression. Interestingly, the supplementation of GSH changed the cell death type from autophagic cell death to apoptotic cell death. Pre-treatment with GSH plus p38 MAP kinase inhibitor (SB203580) strongly diminished the eupalinin A-induced autophagic cell death compared with GSH pre-treatment, suggesting a negative regulation of p38 MAP kinase in this cell death type conversion. Taken together, intracellular ROS levels, including GSH, are crucial for the susceptibility to cell death and the determination of type of eupalinin A-induced cell death.

Effects of sesquiterpene lactones on melanogenesis in mouse B16 melanoma cells.

In this study, we examined the effect of sesquiterpene lactones isolated from Calea urticifolia and Tanacetum parthenium (feverfew) on melanogenesis in mouse B16 melanoma cells. In response to 3-isobutyl-1-methylxanthin (IBMX), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. Treatment of sesquiterpene lactones at lower concentration (1 microM) significantly blocked IBMX-induced melanogenesis, but did not induce the inhibitory activity of cell growth. Among them, 2,3-epoxyjuanislamin exhibited a potent inhibitory effect on melanogenesis. Treatment of B16 cells with 2,3-epoxyjuanislamin elicited significant decreases in tyrosinase protein and mRNA levels. These results demonstrated that the inhibitory effects of sesquiterpene lactones on melanin biosynthesis may be due to the suppression of tyrosinase expression.

Inhibitory effects of polymethoxy flavones isolated from Citrus reticulate on degranulation in rat basophilic leukemia RBL-2H3: enhanced inhibition by their combination.

Polymethoxy flavones (PMFs) are present in fruit tissues of Citrus species. It has been reported that flavonoids isolated from several Citrus have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we examined the effect of PMFs (PMF-1: 6,7,4',5'-tetramethoxy-5-monohydroxyflavone, PMF-2: 5,6,8,3',6'-pentamethoxy flavone, PMF-3: 5,6,7,3',4',5'-hexamethoxy flavone) on the degranulation in RBL-2H3 cells. All the PMFs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. Interestingly, PMF-combination (PMF-1+PMF-2; PMF-1+PMF-3) treatment enhanced the inhibition of degranulation compared with PMF-single treatment. In order to clarify the inhibitory mechanism of degranulation by PMFs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas. All the PMFs significantly suppressed the activation of Syk and PLCgammas. In Ag-mediated activation of Fc epsilonRI on mast cells, three major subfamilies of mitogen-activated protein kinases, especially ERK44/42, were activated. These PMFs reduced the level of phospho-ERKs. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, and PMF treatment reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these PMFs mainly is due to the Syk/PLCgammas/PKC pathway and Ca(2+) influx. Furthermore, to be noted in the PMF-combination treatment, inactivation of Syk was enhanced compared with PMF-single treatment. But the inhibitory effect of degranulation by PMF-combination treatment was not associated with the suppression of Ca(2+) influx.

Inhibitory effects of whisky congeners on melanogenesis in mouse B16 melanoma cells.

We examined the effect of whisky congeners, substances other than ethanol in whisky, on melanogenesis in mouse B16 melanoma cells. Treatment with whisky congeners significantly blocked melanogenesis. Our results indicate that the inhibitory effects of whisky congeners on melanogenesis is due to direct inhibition of tyrosinase activity and to suppression of tyrosinase protein levels.

Inhibitory effect of xanthones isolated from the pericarp of Garcinia mangostana L. on rat basophilic leukemia RBL-2H3 cell degranulation.

Mangostin, Garcinia mangostana L. is used as a traditional medicine in southeast Asia for inflammatory and septic ailments. Hitherto we indicated the anticancer activity induced by xanthones such as alpha-, beta-, and gamma-mangostin which were major constituents of the pericarp of mangosteen fruits. In this study, we examined the effect of xanthones on cell degranulation in rat basophilic leukemia RBL-2H3 cells. Antigen (Ag)-mediated stimulation of high affinity IgE receptor (FcepsilonRI) activates intracellular signal transductions resulting in the release of biologically active mediators such as histamine. The release of histamine and other inflammatory mediators from mast cell or basophils is the primary event in several allergic responses. These xanthones suppressed the release of histamine from IgE-sensitized RBL-2H3 cells. In order to reveal the inhibitory mechanism of degranulation by xanthones, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas. All the xanthones tested significantly suppressed the signaling involving Syk and PLCgammas. In Ag-mediated activation of FcepsilonRI on mast cells, three major subfamilies of mitogen-activated protein kinases were activated. The xanthones decreased the level of phospho-ERKs. Furthermore, the levels of phospho-ERKs were observed to be regulated by Syk/LAT/Ras/ERK pathway rather than PKC/Raf/ERK pathway, suggesting that the inhibitory mechanism of xanthones was mainly due to suppression of the Syk/PLCgammas/PKC pathway. Although intracellular free Ca(2+) concentration ([Ca(2+)](i)) was elevated by FcepsilonRI activation, it was found that alpha- or gamma-mangostin treatment was reduced the [Ca(2+)](i) elevation by suppressed Ca(2+) influx.

Interactive effects of polymethoxy flavones from Citrus on cell growth inhibition in human neuroblastoma SH-SY5Y cells.

Much evidence indicates that typical phytochemicals such as resveratrol, epigallocatechin gallate, and curcumin have a growth inhibitory effect against cancer cells when each is tested separately. However, when fruits and vegetables including a mixture of phytochemicals are consumed, it is unclear whether this anti-proliferative activity is elicited in the body. Initially, we found that nobiletin, a typical polymethoxy flavone from Citrus, had a preventive effect on H(2)O(2)-induced apoptosis at 20-30 microM in human neuroblastoma SH-SY5Y cells. Nobiletin acted as a signal modulator to attenuate the activation of the intrinsic pathway of the apoptosis induced by H(2)O(2) exposure. On the other hand, tangeretin and 5-demethyl nobiletin, which are also polymethoxy flavones from Citrus, were shown to have a growth inhibitory effect by us and others. These results led us to investigate the interactive effects of these polymethoxy flavones on cell growth. In the present study, we found that tangeretin, nobiletin, and 5-demethyl nobiletin exhibited a cancelling, synergistic, or additive effect when combinations of two of these three compounds were tested. As to the structure-activity relationship, the methyl group at C-5 in nobiletin was shown to contribute to the anti-proliferative effect. By the combined treatment with tangeretin and 5-demethyl nobiletin, the apoptotic cell population and the activity of caspase-3 were synergistically elevated. The finding that tangeretin and 5-demethyl nobiletin induced apoptosis by reducing the mitochondrial membrane potential suggested that an intrinsic pathway of apoptosis was synergistically activated by the combination treatment with tangeretin and 5-demethyl nobiletin. On the other hand, in the combined treatment including nobiletin, the growth inhibitory activity of tangeretin was reduced. These results indicate the relevance of the combination of phytochemicals for the enhancement of the anticancer effect.

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway.

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca2+-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca2+ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIdelta by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIdelta and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.

Eupalinin A isolated from Eupatorium chinense L. induces autophagocytosis in human leukemia HL60 cells.

Eupalinin A, a natural phytoalexin included in Eupatorium chinense L., exhibited a marked inhibitory effect on cell growth in HL60 cells. The morphological aspects of eupalinin A-treated cells evaluated by Hoechst 33342 nuclear staining indicated cell death, only a small part of which showed a typical apoptosis with nuclear fragmentation and condensation. To determine what type of cell death is caused by eupalinin A, we examined the contribution of caspases, Bcl-2 family proteins, MAP kinase, and PI3K/Akt, and mitochondrial membrane potential to this cell death. As a result, most part of the cell death was not associated with apoptosis because of caspase independence and no death factor released from mitochondria. Electron microscopic study indicated a characteristic finding of autophagy such as the formation of autophagosomes. Furthermore, the level of microctubule-associated-protein light chain 3 (LC3) II protein and monodancylcanaverin (MDC) incorporation were gradually increased with reduction of mitochondrial membrane potential by the accumulation of intracellular ROS after eupalinin A treatment. From these results, we can conclude that eupalinin A-induced cell death was mainly due to autophagy, which was initiated by increased ROS, resulting in the perturbation of mitochondrial membrane potential. Since the class III PI3K inhibitor such as 3-MA or LY294002 did not inhibit the eupalinin A-induced type II programmed cell death (PCD II), it was suggested that the PCD II was executed by Beclin-1 independent pathway of damage-induced mitochondrial autophagy (mitophagy).

N-trans-feruloyltyramine as a melanin biosynthesis inhibitor.

In this study, we examined the effect of N-trans-feruloyltyramine (FA) on melanogenesis in mouse B16 melanoma cells. Melanogenesis was inhibited by FA in a dose-dependent manner. FA exhibited a greater potency than kojic acid as a standard inhibitor of melanogenesis. Moreover, treatment of B16 melanoma cells with FA was found to cause marked decreases in the expression levels of tyrosinase. FA-induced downregulation of tyrosinase resulted in suppression of melanin biosynthesis in murine B16 melanoma cells.

Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

(-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

Magnolol-induced apoptosis is mediated via the intrinsic pathway with release of AIF from mitochondria in U937 cells.

Magnolol has been reported to have an inhibitory effect on tumor invasion in vitro and in vivo. In this study, we found that treatment with 30 microM magnolol exhibited growth inhibition partly by inducing apoptosis in cultured human leukemia U937 cells and that the apoptosis was induced via the sequential ordering of molecular events; 1) a transient decrease of phosphorylated extracelluar signal-requlated kinase (ERK), 2) translocation of apoptosis inducing factor (AIF) from mitochondria to cytosol concurrent with a decreased membrane potential, and 3) downregulation of bcl-2 protein. Pretreatment of the cells with a pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) did not prevent the apoptosis induced by magnolol. These findings indicated that the above-mentioned sequence of intracellular signaling events led to apoptosis in magnolol-treated U937 cells, which was caspase-independent.