Xanthohumol, a prenylated chalcone, regulates lipid metabolism by modulating the LXRα/RXR-ANGPTL3-LPL axis in hepatic cell lines and high-fat diet-fed zebrafish models.

Angiopoietin-like 3 (ANGPTL3) acts as an inhibitor of lipoprotein lipase (LPL), impeding the breakdown of triglyceride-rich lipoproteins (TGRLs) in circulation. Targeting ANGPTL3 is considered a novel strategy for improving dyslipidemia and atherosclerotic cardiovascular diseases (ASCVD). Hops (Humulus lupulus L.) contain several bioactive prenylflavonoids, including xanthohumol (Xan), isoxanthohumol (Isoxan), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN), with the potential to manage lipid metabolism. The aim of this study was to investigate the lipid-lowering effects of Xan, the effective prenylated chalcone in attenuating ANGPTL3 transcriptional activity, both in vitro using hepatic cells and in vivo using zebrafish models, along with exploring the underlying mechanisms. Xan (10 and 20 µM) significantly reduced ANGPTL3 mRNA and protein expression in HepG2 and Huh7 cells, leading to a marked decrease in secreted ANGPTL3 proteins via hepatic cells. In animal studies, orally administered Xan significantly alleviated plasma triglyceride (TG) and cholesterol levels in zebrafish fed a high-fat diet. Furthermore, it reduced hepatic ANGPTL3 protein levels and increased LPL activity in zebrafish models, indicating its potential to modulate lipid profiles in circulation. Furthermore, molecular docking results predicted that Xan exhibits a higher binding affinity to interact with liver X receptor α (LXRα) and retinoic acid X receptor (RXR) than their respective agonists, T0901317 and 9-Cis-retinoic acid (9-Cis-RA). We observed that Xan suppressed hepatic ANGPTL3 expression by antagonizing the LXRα/RXR-mediated transcription. These findings suggest that Xan ameliorates dyslipidemia by modulating the LXRα/RXR-ANGPTL3-LPL axis. Xan represents a novel potential inhibitor of ANGPTL3 for the prevention or treatment of ASCVD.

8-Hydroxydaidzein Induces Apoptosis and Inhibits AML-Associated Gene Expression in U-937 Cells: Potential Phytochemical for AML Treatment.

BACKGROUND: 8-hydroxydaidzein (8-OHD) is a compound derived from daidzein, known for its anti-inflammatory and anti-proliferative properties in K562 human chronic myeloid leukemia (CML) cells. However, its effects on acute myeloid leukemia (AML) cells have not been fully understood. METHOD: To investigate its potential anti-AML mechanism, we employed an integrated in vitro-in silico approach. RESULTS: Our findings demonstrate that 8-OHD suppresses the expression of CDK6 and CCND2 proteins and induces cell apoptosis in U-937 cells by activating Caspase-7 and cleaving PARP-1. Microarray analysis revealed that 8-OHD downregulates differentially expressed genes (DEGs) associated with rRNA processing and ribosome biogenesis pathways. Moreover, AML-target genes, including CCND2, MYC, NPM1, FLT3, and TERT, were downregulated by 8-OHD. Additionally, molecular docking software predicted that 8-OHD has the potential to interact with CDK6, FLT3, and TERT proteins, thereby reducing their activity and inhibiting cell proliferation. Notably, we discovered a synergic pharmacological interaction between 8-OHD and cytarabine (Ara-C). CONCLUSIONS: Overall, this study provides insights into the therapeutic applications of 8-OHD in treating AML and elucidates its underlying mechanisms of action.

5-Demethylnobiletin Inhibits Cell Proliferation, Downregulates ID1 Expression, Modulates the NF-κB/TNF-α Pathway and Exerts Antileukemic Effects in AML Cells.

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20−80 µM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 µM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 µM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 µM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

8-Hydroxydaidzein Downregulates JAK/STAT, MMP, Oxidative Phosphorylation, and PI3K/AKT Pathways in K562 Cells.

A metabolite isolated from fermented soybean, 8-hydroxydaidzein (8-OHD, 7,8,4'-trihydroxyisoflavone, NSC-678112), is widely used in ethnopharmacological research due to its anti-proliferative and anti-inflammatory effects. We reported previously that 8-OHD provoked reactive oxygen species (ROS) overproduction, and induced autophagy, apoptosis, breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) degradation, and differentiation in K562 human chronic myeloid leukemia (CML) cells. However, how 8-OHD regulates metabolism, the extracellular matrix during invasion and metastasis, and survival signaling pathways in CML remains largely unexplored. High-throughput technologies have been widely used to discover the therapeutic targets and pathways of drugs. Bioinformatics analysis of 8-OHD-downregulated differentially expressed genes (DEGs) revealed that Janus kinase/signal transducer and activator of transcription (JAK/STAT), matrix metalloproteinases (MMPs), c-Myc, phosphoinositide 3-kinase (PI3K)/AKT, and oxidative phosphorylation (OXPHOS) metabolic pathways were significantly altered by 8-OHD treatment. Western blot analyses validated that 8-OHD significantly downregulated cytosolic JAK2 and the expression and phosphorylation of STAT3 dose- and time-dependently in K562 cells. Zymography and transwell assays also confirmed that K562-secreted MMP9 and invasion activities were dose-dependently inhibited by 8-OHD after 24 h of treatment. RT-qPCR analyses verified that 8-OHD repressed metastasis and OXPHOS-related genes. In combination with DisGeNET, it was found that 8-OHD's downregulation of PI3K/AKT is crucial for controlling CML development. A STRING protein-protein interaction analysis further revealed that AKT and MYC are hub proteins for cancer progression. Western blotting revealed that AKT phosphorylation and nuclear MYC expression were significantly inhibited by 8-OHD. Collectively, this systematic investigation revealed that 8-OHD exerts anti-CML effects by downregulating JAK/STAT, PI3K/AKT, MMP, and OXPHOS pathways, and MYC expression. These results could shed new light on the development of 8-OHD for CML therapy.

The Lipid-Modulating Effect of Tangeretin on the Inhibition of Angiopoietin-like 3 (ANGPTL3) Gene Expression through Regulation of LXRα Activation in Hepatic Cells.

The excessive accumulation of TG-rich lipoproteins (TGRLs) in plasma is associated with dyslipidemia and atherosclerotic cardiovascular diseases (ASCVDs). Tangeretin is a bioactive pentamethoxyflavone mainly found in citrus peels, and it has been reported to protect against hyperlipidemia, diabetes, and obesity. The aim of this study was to investigate the lipid-modulating effects and the underlying mechanisms of tangeretin action in hepatic cells. Transcriptome and bioinformatics analyses with the Gene Ontology (GO) database showed that tangeretin significantly regulated a set of 13 differentially expressed genes (DEGs) associated with the regulation of lipoprotein lipase (LPL) activity. Among these DEGs, angiopoietin-like 3 (ANGPTL3), an essential inhibitor of LPL catalytic activity that regulates TGRL metabolism in plasma, was markedly downregulated by tangeretin. We demonstrated that tangeretin significantly inhibited the mRNA expression of ANGPTL3 in HepG2 and Huh-7 cells. Tangeretin treatment of hepatic cells also reduced the levels of both intracellular and secreted ANGPTL3 proteins. Moreover, we found that inhibition of ANGPTL3 production by tangeretin augmented LPL activity. We further demonstrated that the transcriptional activity of the ANGPTL3 promoter was significantly attenuated by tangeretin, and we identified a DNA element located between the -250 and -121 positions that responded to tangeretin. Furthermore, we found that tangeretin did not alter the levels of the nuclear liver X receptor α (LXRα) protein, an essential transcription factor that binds to the tangeretin-responsive element, but it can counteract LXRα-mediated ANGPTL3 transcription. On the basis of molecular docking analysis, tangeretin was predicted to bind to the ligand-binding domain of LXRα, which would result in suppression of LXRα activation. Our findings support the hypothesis that tangeretin exerts a lipid-lowering effect by modulating the LXRα-ANGPTL3-LPL pathway, and thus, it can be used as a potential phytochemical for the prevention or treatment of dyslipidemia.

8-Hydroxydaidzein, an Isoflavone from Fermented Soybean, Induces Autophagy, Apoptosis, Differentiation, and Degradation of Oncoprotein BCR-ABL in K562 Cells.

8-Hydroxydaidzein (8-OHD, 7,8,4'-trihydoxyisoflavone) is a hydroxylated derivative of daidzein isolated from fermented soybean products. The aim of this study is to investigate the anti-proliferative effects and the underlying mechanisms of 8-OHD in K562 human chronic myeloid leukemia (CML) cells. We found that 8-OHD induced reactive oxygen species (ROS) overproduction and cell cycle arrest at the S phase by upregulating p21Cip1 and downregulating cyclin D2 (CCND2) and cyclin-dependent kinase 6 (CDK6) expression. 8-OHD also induced autophagy, caspase-7-dependent apoptosis, and the degradation of BCR-ABL oncoprotein. 8-OHD promoted Early Growth Response 1 (EGR1)-mediated megakaryocytic differentiation as an increased expression of marker genes, CD61 and CD42b, and the formation of multi-lobulated nuclei in enlarged K562 cells. A microarray-based transcriptome analysis revealed a total of 3174 differentially expressed genes (DEGs) after 8-OHD (100 µM) treatment for 48 h. Bioinformatics analysis of DEGs showed that hemopoiesis, cell cycle regulation, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducers and activators of transcription (JAK-STAT)-mediated apoptosis/anti-apoptosis networks were significantly regulated by 8-OHD. Western blot analysis confirmed that 8-OHD significantly induced the activation of MAPK and NF-κB signaling pathways, both of which may be responsible, at least in part, for the stimulation of apoptosis, autophagy, and differentiation in K562 cells. This is the first report on the anti-CML effects of 8-OHD and the combination of experimental and in silico analyses could provide a better understanding for the development of 8-OHD on CML therapy.

Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown exert anti-inflammatory activities and relieve oxidative stress via Nrf2 activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown have anti-inflammatory properties and are popularly used as complementary and alternative medicine in Asia. AIM OF THE STUDY: To investigate the underlying molecular mechanism and active chemicals in the ethanol extracts of V. patula (VP) and L. chinensis (LC). MATERIALS AND METHODS: The inhibitory activities of VP and LC on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and interleukin-6 (IL-6) production were investigated in RAW264.7 macrophages and BV2 microglia. Downregulation of pro-inflammatory genes and upregulation of Nrf2 (NF-E2 p45-related factor 2)-ARE (antioxidant response element) pathway were investigated using RT-Q-PCR and Western blotting. Direct antioxidant capacities were measured using free radical scavenging and Folin-Ciocalteu assays. The flavonoids and triterpenes in VP and LC were identified by HPLC-ESI-MS. RESULTS: VP and LC inhibited NO and IL-6 production and suppressed iNOS, IL-6, IL-1ß and CCL2 gene expression. VP and LC were potent direct antioxidants and effective indirect antioxidants assayed by Nrf2 activation and induction of heme oxygenase (HO)-1, glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase 1 (NQO1). Three flavonoids including apigenin (1), luteolin (2) and chryseriol (3), and one triterpene betulinic acid (4) were found in VP; while compounds 1-4 and oleanolic acid (5) were in LC. CONCLUSION: Anti-inflammatory and antioxidant activities of VP and LC may be in great part attributed to the identified Nrf2 activating compounds, which induce expression of Phase II enzymes and attenuate the upregulation of pro-inflammatory genes.

Sinomenine Inhibits Migration and Invasion of Human Lung Cancer Cell through Downregulating Expression of miR-21 and MMPs.

Sinomenine is an alkaloid derived from Sinomenium acutum. Recent studies have found that sinomenine can inhibit various cancers by inhibiting the proliferation, migration and invasion of tumors and inducing apoptosis. This study aims to investigate the effect and mechanism of sinomenine on inhibiting the migration and invasion of human lung adenocarcinoma cells in vitro. The results demonstrate that viabilities of A549 and H1299 cells were inhibited by sinomenine in a dose-dependent manner. When treated with sub-toxic doses of sinomenine, cell migration and invasion are markedly suppressed. Sinomenine decreases the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9, and the extracellular inducer of matrix metalloproteinase (EMMPRIN/CD147), but elevates the expression of reversion-inducing cysteine-rich proteins with kazal motifs (RECK) and the tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. In addition, sinomenine significantly increases the expression of the epithelial marker E-cadherin but concomitantly decreases the expression of the mesenchymal marker vimentin, suggesting that it suppresses epithelial-mesenchymal transition (EMT). Moreover, sinomenine downregulates oncogenic microRNA-21 (miR-21), which has been known to target RECK. The downregulation of miR-21 decreases cell invasion, while the upregulation of miR-21 increases cell invasion. Furthermore, the downregulation of miR-21 stimulates the expression of RECK, TIMP-1/-2, and E-cadherin, but reduces the expression of MMP-2/-9, EMMPRIN/CD147, and vimentin. Taken together, the results reveal that the inhibition of A549 cell invasion by sinomenine may, at least in part, be through the downregulating expression of MMPs and miR-21. These findings demonstrate an attractive therapeutic potential for sinomenine in lung cancer anti-metastatic therapy.

Nobiletin Promotes Megakaryocytic Differentiation through the MAPK/ERK-Dependent EGR1 Expression and Exerts Anti-Leukemic Effects in Human Chronic Myeloid Leukemia (CML) K562 Cells.

Differentiation therapy is an alternative strategy used to induce the differentiation of blast cells toward mature cells and to inhibit tumor cell proliferation for cancer treatment. Nobiletin (NOB), a polymethoxyflavone phytochemical, is present abundantly in citrus peels and has been reported to possess anti-cancer activity. In this study, we investigated the anti-leukemic effects of NOB on cell differentiation and its underlying mechanisms in human chronic myeloid leukemia (CML) K562 cells. NOB (100 µM) treatment for 24 and 48 h significantly decreased viability of K562 cells to 54.4 ± 5.3% and 46.2 ± 9.9%, respectively. NOB (10-100 µM) significantly inhibited cell growth in K562 cells. Flow cytometry analysis and immunoblotting data showed that NOB (40 and 80 µM) could modulate the cell cycle regulators including p21, p27, and cyclin D2, and induce G1 phase arrest. NOB also increased the messenger RNA (mRNA) and protein expression of megakaryocytic differentiation markers, such as CD61, CD41, and CD42 as well as the formation of large cells with multi-lobulated nuclei in K562 cells. These results suggested that NOB facilitated K562 cells toward megakaryocytic differentiation. Furthermore, microarray analysis showed that expression of EGR1, a gene associated with promotion of megakaryocytic differentiation, was markedly elevated in NOB-treated K562 cells. The knockdown of EGR1 expression by small interference RNA (siRNA) could significantly attenuate NOB-mediated cell differentiation. We further elucidated that NOB induced EGR1 expression and CD61 expression through increases in MAPK/ERK phosphorylation in K562 cells. These results indicate that NOB promotes megakaryocytic differentiation through the MAPK/ERK pathway-dependent EGR1 expression in human CML cells. In addition, NOB when combined with imatinib could synergistically reduce the viability of K562 cells. Our findings suggest that NOB may serve as a beneficial anti-leukemic agent for differentiation therapy.

Reinforcement of Tobacco Control and Reduction in Medical Utilization for Asthma in Taiwan: A Population-Based Study.

Environmental air quality can affect asthma control and the development of overt asthmatic manifestations. In this population-based study, we investigated the effect of reinforcing a smoking ban in Taiwan through the enactment of the Tobacco Hazards and Prevention Act (THPA) on healthcare utilization rate by asthmatics. Analysis was performed based on data relevant to non-hospitalized asthmatic patients with insurance claims between 2005 and 2013 from the National Health Insurance Research Database of Taiwan, reported data on Asian dust storms, and penalty rates for violations of the tobacco ban. Poisson regression showed that the risk for outpatient visits for asthma was lower after enactment of the THPA (RR = 0.98, 95% CI = 0.98-0.99), with a yearly trend of a reduced risk (RR = 0.99, 95% CI = 0.99-1.00), also lower in geographic regions with medium (RR = 0.79, 95% CI = 0.79-0.80) and high (RR = 0.91, 95% CI = 0.91-0.92) penalty rates. Subgroup analysis showed that asthma visit rates were reduced in both male and female groups after the enactment of the THPA. The risk of an asthma ER visit was increased after the enactment of the amended THPA (RR = 1.07, 95% CI = 1.05-1.09), although the yearly trend was not significant (RR = 1.00, 95% CI = 1.00-1.00). The risk of emergency room visits for asthma was significantly reduced in regions with medium (RR = 0.68, 95% CI = 0.68-0.69) and high (RR = 0.75, 95% CI = 0.74-0.76) penalty rates. Subgroup analysis showed that the visit rates were similar in both male and female groups. The effectiveness of reinforcing the smoking ban warrants further policies aimed at further reducing passive smoking.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Nobiletin Down-Regulates c-KIT Gene Expression and Exerts Antileukemic Effects on Human Acute Myeloid Leukemia Cells.

Nobiletin, a dietary citrus flavonoid, has been reported to possess several biological activities such as antioxidant, anti-inflammatory, and anticancer properties. The aim of this study was to investigate the antileukemic effects of nobiletin and its underlying mechanisms on human acute myeloid leukemia (AML) cells. We demonstrated that nobiletin (0-100 µM) significantly reduced cell viability from 100.0 ± 9.6% to 31.1 ± 2.8% in human AML THP-1 cell line. Nobiletin arrested cell cycle progression in G1 phase and induced myeloid cell differentiation in human AML cells. Microarray analysis showed that mRNA expression of the c- KIT gene, a critical proto-oncogene associated with leukemia progression, was dramatically reduced in nobiletin-treated AML cells. Furthermore, we verified that AML cells treated with nobiletin (40 and 80 µM) for 48 h markedly suppressed c-KIT mRNA expression (from 1.00 ± 0.07-fold to 0.62 ± 0.08- and 0.30 ± 0.05-fold) and reduced the level of c-KIT protein expression (from 1.00 ± 0.11-fold to 0.60 ± 0.15- and 0.34 ± 0.05-fold) by inhibition of KIT promoter activity. The knockdown of c-KIT expression by shRNA attenuated cancer cell growth and induced cell differentiation. Moreover, we found that the overexpression of c-KIT abolished nobiletin-mediated cell growth inhibition in leukemia cells. These results indicate that nobiletin exerts antileukemic effects through the down-regulation of c-KIT gene expression in AML cells. Finally, we demonstrated that the combination of a conventional AML chemotherapeutic agent, cytarabine, with nobiletin resulted in more reduction of cell viability in AML cells. Our current findings suggest that nobiletin is a novel c-KIT inhibitor and may serve as a chemo-preventive or -therapeutic agent against human AML.

Pinostrobin Inhibits Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) Gene Expression through the Modulation of FoxO3a Protein in HepG2 Cells.

Pinostrobin, a flavonoid phytochemical found in variety of plants, has been demonstrated to possess numerous bioactivities such as antioxidant, anti-inflammatory, anticancer, and neuroprotective properties. The aim of this study was to investigate the hypocholesterolemic effect of pinostrobin on the regulation of the gene expression of PCSK9 and its underlying mechanisms in hepatic cells. We found that pinostrobin (20 and 40 µM) significantly inhibited the PCSK9 promoter activity from 1.00 ± 0.16 (fold) to 0.85 ± 0.06 and 0.54 ± 0.05, respectively, as well as the suppression of PCSK9 mRNA expression from 1.00 ± 0.11 (fold) to 0.81 ± 0.07 and 0.58 ± 0.07, respectively, in HepG2 cells. Pinostrobin significantly reduced the mature form of the PCSK9 protein, inhibited the catalytic activity of PCSK9, and increased the protein level of LDLR and the LDL uptake activity in HepG2 cells. We further demonstrated that pinostrobin markedly increased the level of nuclear forkhead box O3a (FoxO3a) protein, enhanced FoxO3a/PCSK9 promoter complexes formation, and attenuated the promoter binding capacity of nuclear HNF-1α. The knockdown of FoxO3a in HepG2 cells by small interference RNA (siRNA) abolished the pinostrobin-mediated PCSK9 reduction. Finally, we demonstrated that pinostrobin attenuated simvastatin-induced PCSK9 overexpression in HepG2 cells. Our current findings reveal that pinostrobin is a PCSK9 inhibitor and down-regulates the PCSK9 gene expression through the up-regulation of the FoxO3a level in hepatic cells. Pinostrobin with potential PCSK9 inhibitory activity may serve as a novel agent for cholesterol regulation and lipid management.

Anti-inflammatory Activity of 8-Hydroxydaidzein in LPS-Stimulated BV2 Microglial Cells via Activation of Nrf2-Antioxidant and Attenuation of Akt/NF-κB-Inflammatory Signaling Pathways, as Well As Inhibition of COX-2 Activity.

It was demonstrated that isoflavones can cross the blood-brain barrier, making them desirable candidate agents for the prevention of neurological symptoms. 8-Hydroxydaidzein (8-OHD, 4',7,8-trihydoxyisoflavone) is an isoflavone found only in fermented soy food. Current results showed that 8-OHD inhibited LPS-stimulated production of nitric oxide (NO) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, by inhibiting gene expression in BV2 microglial cells. Moreover, 8-OHD markedly quenched reactive oxygen species (ROS) and activated NF-E2-related factor 2 (Nrf2) so as to upregulate expression of Phase II enzymes, including heme oxygenase (HO)-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and the modifier subunit of glutamate cysteine ligase (GCLM). 8-OHD also suppressed LPS-stimulated phosphorylation of Akt and NF-κB-p65. The anti-inflammatory activity of 8-OHD was attenuated by the HO-1 inhibitor zinc protoporphyrin (Znpp) but augmented by the PI3K/Akt inhibitor LY294002. 8-OHD also diminished LPS-induced prostaglandin E2 (PGE2) production without affecting cyclooxygenase (COX)-2 expression. In vitro assay shows that 8-OHD displayed mixed-type inhibition of COX-2 with an IC50 of 8.9 ± 1.2 µM. These data suggest that the anti-inflammatory activity of 8-OHD may be associated with the activation of Nrf2/HO-1 and attenuation of Akt/NF-κB signaling pathways as well as inhibition of COX-2 enzyme activity. In conclusion, 8-OHD, a potent Nrf2 activator, Akt/NF-κB activation suppressor, and COX-2 enzyme inhibitor, may have health-promoting effects for mitigating microglia activation and preventing neuroinflammation.

Response of Myeloid Leukemia Cells to Luteolin is Modulated by Differentially Expressed Pituitary Tumor-Transforming Gene 1 (PTTG1) Oncoprotein.

Luteolin, a flavonoid nutraceutical abundant in vegetables and fruits, exhibits a wide range of bioactive properties, including antioxidant, anti-inflammatory and anti-cancer activities. Pituitary tumor-transforming gene 1 (PTTG1), an oncoprotein that regulates cell proliferation, is highly expressed in several types of cancer cells including leukemia. In this study, we aim to investigate the anti-cancer effects of luteolin on cells with differential PTTG1 expression and their underlying mechanisms in human myeloid leukemia cells. Methyl thiazolyl tetrazolium (MTT) assay data showed that luteolin (25-100 µM) significantly reduced cell viability in THP-1, HL-60 and K562 cells but did not affect normal peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis and Western blot data demonstrated that luteolin induced a stronger apoptosis on undifferentiated myeloid leukemia cells with higher PTTG1 protein levels than on 12-myristate 13-acetate (PMA)- or all-trans-retinoic acid (ATRA)-differentiated cells with lower PTTG1 expression. Furthermore, PTTG1 knockdown by shRNA in leukemia cells suppressed cell proliferation, arrested cell-cycle progression and impaired the effectiveness of luteolin on cell-cycle regulation. Moreover, PTTG1-knockdown cells with luteolin exposure presented a reduction of the apoptotic proteins and maintained higher levels of the anti-apoptotic proteins such as Mcl-1, Bcl-2 and p21, which exhibited greater resistance to apoptosis. Finally, microarray analysis showed that 20 genes associated with cell proliferation, such as CXCL10, VEGFA, TNF, TP63 and FGFR1, were dramatically down-regulated in PTTG1-knockdown cells. Our current findings clearly demonstrate that luteolin-triggered leukemic cell apoptosis is modulated by the differential expression of the PTTG1. PTTG1 oncoprotein overexpression may modulate cell proliferation-related regulators and enhance the response of myeloid leukemia cells to luteolin. Luteolin is beneficial for the treatment of cancer cells with highly expressed PTTG1 oncoprotein.

Xanthohumol Suppresses Mylip/Idol Gene Expression and Modulates LDLR Abundance and Activity in HepG2 Cells.

Xanthohumol, a prenylated flavonoid found in hops (Humulus lupulus L.), exhibits multiple biological activities such as antiatherosclerosis and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effects and molecular mechanisms of xanthohumol in hepatic cells. We found that xanthohumol (10 and 20 µM) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) from 100.0 ± 2.1% to 115.0 ± 1.3% and 135.2 ± 2.7%, and enhanced the LDL uptake activity from 100.0 ± 0.9% to 139.1 ± 13.2% in HepG2 cells (p < 0.01). The mRNA levels of LDLR, HMGCR, and PCSK9 were not altered. Xanthohumol (20 µM) reduced the expression of inducible degrader of the LDL receptor (Mylip/Idol) mRNA and protein by approximately 45% (p < 0.01), which was reported to be associated with increases of LDLR level. We demonstrated that xanthohumol suppressed hepatic Mylip/Idol expression via counteracting liver X receptor (LXR) activation. The molecular docking results predicted that xanthohumol has a high binding affinity to interact with the LXRα ligand-binding domain, which may result in attenuation of LXRα-induced Mylip/Idol expression. Finally, we demonstrated that the Mylip/Idol expression and LDLR activity were synergistically changed by a combination of xanthohumol and simvastatin treatment. Our findings indicated that xanthohumol may regulate the LXR-Mylip/Idol axis to modulate hepatic LDLR abundance and activity.

Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells.

BACKGROUND: Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. METHODS: Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. RESULTS: Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity.

Solasodine inhibits invasion of human lung cancer cell through downregulation of miR-21 and MMPs expression.

Solasodine, a naturally occurring aglycone of glycoalkaloid in eggplant (Solanum melongena), was found to inhibit proliferation in various tumor cells. However, the effect of solasodine on cancer cell metastasis remains unclear. This study investigates the suppression mechanism of solasodine on motility of human lung cancer cell A549 in vitro. Results show that solasodine reduces viability of A549 cells. Treatment with non-toxic doses of solasodine suppresses markedly cell invasion. Solasodine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9 and extracellular inducer of matrix metalloproteinase (EMMPRIN), but increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK), as well as tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Immunoblotting assays indicate that solasodine is effective in suppressing PI3K and Akt phosphorylation. Moreover, solasodine downregulates oncogenic microRNA-21 (miR-21), which has been known to target RECK. Downregulation of miR-21 by miR-21 inhibitor increases RECK expression and decreases cell invasion, suggesting that downregulation of miR-21 by solasodine may contribute to elevate RECK expression and subsequently inhibiting cell invasion. Taken together, the results reveal that inhibition of A549 cell invasion by solasodine may be, at least in part, through blocking MMP expression. Solasodine also reduces PI3K/Akt signaling pathways and downregulates expression of miR-21. These findings demonstrate an attractive therapeutic potential for solasodine in lung cancer anti-metastatic therapy.

Tanshinone IIA Modulates Low Density Lipoprotein Uptake via Down-Regulation of PCSK9 Gene Expression in HepG2 Cells.

Tanshinone IIA, one of the most pharmacologically bioactive phytochemicals isolated from Salvia miltiorrhiza Bunge, possesses several biological activities such as anti-inflammation, anti-cancer, neuroprotection and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effect of tanshinone IIA in hepatic cells. We demonstrated that tanshinone IIA significantly increased the amount of low-density lipoprotein receptor (LDLR) and LDL uptake activity in HepG2 cells at the post-transcriptional regulation. We further demonstrated that tanshinone IIA inhibited the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and mature protein, which may lead to an increase the cell-surface LDLR in hepatic cells. We further identified a regulatory DNA element involved in the tanshinone IIA-mediated PCSK9 down-regulation, which is located between the -411 and -336 positions of the PCSK9 promoter. Moreover, we found that tanshinone IIA markedly increased the nuclear forkhead box O3a (FoxO3a) level, enhanced FoxO3a/PCSK9 promoter complexes formation and decreased the PCSK9 promoter binding capacity of hepatocyte nuclear factor 1α (HNF-1α), resulting in suppression of PCSK9 gene expression. Finally, we found that the statin-induced PCSK9 overexpression was attenuated and the LDLR activity was elevated in a synergic manner by combination of tanshinone IIA treatment in HepG2 cells. Overall, our results reveal that the tanshinone IIA modulates LDLR level and activity via down-regulation of PCSK9 expression in hepatic cells. Our current findings provide a molecular basis of tanshinone IIA to develop PCSK9 inhibitors for cholesterol management.

Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia.

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1ß, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1ß and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti-neuroinflammatory activities by inhibiting pro-inflammatory mediator expression and production, upregulating HO-1, GCLM and NQO1, blocking NF-κB and modulating JNK signaling pathways. They may offer therapeutic potential for suppressing overactivated microglia and alleviating neurodegeneration.