The Citrus Flavonoid Nobiletin Downregulates Angiopoietin-like Protein 3 (ANGPTL3) Expression and Exhibits Lipid-Modulating Effects in Hepatic Cells and Adult Zebrafish Models.

Nobiletin, a dietary citrus flavonoid, exerts biological activities against hyperlipidemia, obesity, and atherosclerotic cardiovascular diseases (ASCVDs). The aim of this study was to explore the lipid-lowering effects of nobiletin and the underlying molecular mechanisms in vitro in hepatic cells and in vivo in zebrafish models. Transcriptome and gene ontology (GO) analyses of differentially expressed genes (DEGs) by gene set enrichment analysis (GSEA) showed that a set of twenty-eight core enrichment DEGs associated with "GO BP regulation of lipid metabolic process" (GO: 0019216) were significantly downregulated in nobiletin-treated cells. Among these genes, angiopoietin-like 3 (ANGPTL3), an inhibitor of lipoprotein lipase (LPL) activity that regulates TG-rich lipoprotein (TGRL) metabolism in circulation, was the protein most markedly downregulated by nobiletin. Nobiletin (20 and 40 µM) significantly reduced the levels of ANGPTL3 mRNA and intracellular and secreted ANGPTL3 proteins in hepatic cell lines. Furthermore, alleviation of secreted ANGPTL3 production by nobiletin was found to reinstate LPL catalytic activity. Nobiletin significantly inhibited ANGPTL3 promoter activity and attenuated the transcription factor liver X receptor-α (LXRα)-mediated ANGPTL3 transcription. Molecular docking analysis predicted that nobiletin could bind to the ligand-binding domain of LXRα, thereby counteracting LXRα activation. In animal studies, orally administered nobiletin significantly alleviated the levels of plasma triglycerides (TGs) and cholesterol in zebrafish fed a high-fat diet. Moreover, nobiletin significantly reduced the amounts of hepatic ANGPTL3 protein in zebrafish. Our findings suggest that nobiletin may regulate the LXRα-ANGPTL3-LPL axis and exhibit lipid-modulating effects in vitro and in vivo. Thus, nobiletin is a potential ANGPTL3 inhibitor for the regulation of lipid metabolism to ameliorate dyslipidemia and ASCVDs.

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.

Angiopoietin-Like Protein 3 (ANGPTL3) Modulates Lipoprotein Metabolism and Dyslipidemia.

Dyslipidemia is characterized by increasing plasma levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides (TGs) and TG-rich lipoproteins (TGRLs) and is a major risk factor for the development of atherosclerotic cardiovascular disorders (ASCVDs). It is important to understand the metabolic mechanisms underlying dyslipidemia to develop effective strategies against ASCVDs. Angiopoietin-like 3 (ANGPTL3), a member of the angiopoietin-like protein family exclusively synthesized in the liver, has been demonstrated to be a critical regulator of lipoprotein metabolism to inhibit lipoprotein lipase (LPL) activity. Genetic, biochemical, and clinical studies in animals and humans have shown that loss of function, inactivation, or downregulated expression of ANGPTL3 is associated with an obvious reduction in plasma levels of TGs, LDL-C, and high-density lipoprotein-cholesterol (HDL-C), atherosclerotic lesions, and the risk of cardiovascular events. Therefore, ANGPTL3 is considered an alternative target for lipid-lowering therapy. Emerging studies have focused on ANGPTL3 inhibition via antisense oligonucleotides (ASOs) and monoclonal antibody-based therapies, which have been carried out in mouse or monkey models and in human clinical studies for the management of dyslipidemia and ASCVDs. This review will summarize the current literature on the important role of ANGPTL3 in controlling lipoprotein metabolism and dyslipidemia, with an emphasis on anti-ANGPTL3 therapies as a potential strategy for the treatment of dyslipidemia and ASCVDs.

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.

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.

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.

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.

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.

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.

Antioxidant, Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria.

Camellia tenuifloria is an indigenous Camellia species used for the production of camellia oil in Taiwan. This study investigated for the first time the potential antioxidant, anti-tyrosinase and anti-inflammatory activities of oil production byproducts, specifically those of the fruit shell, seed shell, and seed pomace from C. tenuifloria. It was found that the crude ethanol extract of the seed shell had the strongest DPPH scavenging and mushroom tyrosinase inhibitory activities, followed by the fruit shell, while seed pomace was the weakest. The IC50 values of crude extracts and fractions on monophenolase were smaller than diphenolase. The phenolic-rich methanol fraction of seed shell (SM) reduced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. It also repressed the expression of IL-1ß, and secretion of prostaglandin E2 (PGE2) and IL-6 in response to LPS. SM strongly stimulated heme oxygenase 1 (HO-1) expression and addition of zinc protoporphyrin (ZnPP), a HO-1 competitive inhibitor, reversed the inhibition of NO production, indicating the involvement of HO-1 in its anti-inflammatory activity. The effects observed in this study provide evidence for the reuse of residues from C. tenuifloria in the food additive, medicine and cosmetic industries.

Arsenic trioxide induces unfolded protein response in vascular endothelial cells.

Chronic arsenic exposure has been linked to endothelial dysfunction and apoptosis. We investigate the involvement of unfolded protein response (UPR) signaling in the arsenic-mediated cytotoxicity of the SVEC4-10 mouse endothelial cells. The SVEC4-10 cells underwent apoptosis in response to As2O3 dose- and time-dependently, accompanied by increased accumulation of calcium, and activation of caspase-3. These phenomena were completely inhibited by α-lipoic acid (LA), which did not scavenge ROS over-production, but were only partially or not ameliorated by tiron, a potent superoxide scavenger. Moreover, arsenic activated UPR, leading to phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2α), induction of ATF4, and processing of ATF6. Treatment with arsenic also triggered the expression of endoplasmic reticulum (ER) stress markers, GRP78 (glucose-regulated protein), and CHOP (C/EBP homologous protein). The activation of eIF2α, ATF4 and ATF6 and expression of GRP78 and CHOP are repressed by both LA and tiron, indicating arsenic-induced UPR is mediated through ROS-dependent and ROS-independent pathways. Arsenic also induced ER stress-inducible genes, BAX, PUMA (p53 upregulated modulator of apoptosis), TRB3 (tribbles-related protein 3), and SNAT2 (sodium-dependent neutral amino acid transporter 2). Consistent with intracellular calcium and cell viability data, ROS may not be important in arsenic-induced death, because tiron did not affect the expression of these pro-apoptotic genes. In addition, pretreatment with salubrinal, a selective inhibitor of eIF2α dephosphorylation, enhanced arsenic-induced GRP78 and CHOP expression and partially prevented arsenic cytotoxicity in SVEC4-10 cells. Taken together, these results suggest that arsenic-induced endothelial cytotoxicity is associated with ER stress, which is mediated by ROS-dependent and ROS-independent signaling.

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.

The Neuroprotective Effects of Flavonoid Fisetin against Corticosterone-Induced Cell Death through Modulation of ERK, p38, and PI3K/Akt/FOXO3a-Dependent Pathways in PC12 Cells.

The overactive hypothalamic-pituitary-adrenal (HPA) axis is believed to trigger the overproduction of corticosterone, leading to neurotoxicity in the brain. Fisetin is a flavonoid commonly found in fruits and vegetables. It has been suggested to possess various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study aims to explore the potential neuroprotective properties of fisetin against corticosterone-induced cell death and its underlying molecular mechanism in PC12 cells. Our results indicate that fisetin, at concentrations ranging from 5 to 40 µM, significantly protected PC12 cells against corticosterone-induced cell death. Fisetin effectively reduced the corticosterone-mediated generation of reactive oxygen species (ROS) in PC12 cells. Fisetin treatments also showed potential in inhibiting the corticosterone-induced apoptosis of PC12 cells. Moreover, inhibitors targeting MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K) were found to significantly block the increase in cell viability induced by fisetin in corticosterone-treated cells. Consistently, fisetin enhanced the phosphorylation levels of ERK, p38, Akt, and c-AMP response element-binding protein (CREB) in PC12 cells. Additionally, it was found that the diminished levels of p-CREB and p-ERK by corticosterone can be restored by fisetin treatment. Furthermore, the investigation of crosstalk between ERK and CREB revealed that p-CREB activation by fisetin occurred through the ERK-independent pathway. Moreover, we demonstrated that fisetin effectively counteracted the corticosterone-induced nuclear accumulation of FOXO3a, an apoptosis-triggering transcription factor, and concurrently promoted FOXO3a phosphorylation and its subsequent cytoplasmic localization through the PI3K/Akt pathway. In conclusion, our findings indicate that fisetin exerts its neuroprotective effect against corticosterone-induced cell death by modulating ERK, p38, and the PI3K/Akt/FOXO3a-dependent pathways in PC12 cells. Fisetin emerges as a promising phytochemical for neuroprotection.

Arsenic modulates heme oxygenase-1, interleukin-6, and vascular endothelial growth factor expression in endothelial cells: roles of ROS, NF-κB, and MAPK pathways.

Chronic arsenic exposure has been linked to an increased risk of vascular diseases. To clarify the molecular mechanisms through which arsenic causes injuries to blood vessels, we analyzed the effects of arsenic trioxide on the cytotoxicity, intracellular reactive oxygen species (ROS), the expression of related genes, and signaling pathways involved in the SVEC4-10 mouse endothelial cells. Arsenic dose-dependently caused SVEC4-10 cell death, which is completely inhibited by α-lipoic acid (LA), a thioreductant, but partially ameliorated by Tiron, a potent superoxide scavenger. The mRNA levels of heme oxygenase-1 (HO-1), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were significantly increased by arsenic. The up-regulation of these can be blocked by LA instead of Tiron, suggesting ROS is not important in their increase. HO-1 competitive inhibitor zinc protoporphyrin improved the cytotoxicity of arsenic in an inverted-U dose-response curve, indicating the biphasic hormetic effect of HO-1. HO-1 siRNA decreased VEGF expression in response to arsenic. Arsenic exposure also enhanced NF-E2-related factor 2 (Nrf2) expression and increased activation of nuclear factor-κB (NF-κB). NF-κB inhibitor Bay 11-7082 reduced arsenic-mediated expression of HO-1 and IL-6. Selective blocking of the MAPK pathways with p38 inhibitor SB203580 significantly decreased arsenic-induced HO-1 and VEGF expression, while JNKs inhibitor SP600125 increased IL-6 expression. These results suggest that in arsenic-treated SVEC4-10 cells, HO-1 expression is mediated through Nrf2-, NF-κB-, and p38 MAPK-dependent signaling pathways and serves as an upstream regulator of VEGF. IL-6 expression is regulated by NF-κB and JNKs. In conclusion, oxidative stress may be associated with arsenic-induced cytotoxicity and endothelial gene up-regulation, but signaling transduction dominates the direct effects of ROS.

Nobiletin metabolite, 3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone, inhibits LDL oxidation and down-regulates scavenger receptor expression and activity in THP-1 cells.

There is accumulating evidence that LDL oxidation is essential for atherogenesis and antioxidants that prevent oxidation may either decelerate or reduce atherogenesis. Current study focused on the effect and mechanism of 3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone (DTF), a major metabolite of nobiletin (NOB, a citrus polymethoxylated flavone) on atherogenesis. We found DTF had stronger inhibitory activity than alpha-tocopherol on inhibiting Cu2+-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. Monocyte-to-macrophage differentiation plays a vital role in early atherogenesis. DTF (10-20 microM) dose-dependently attenuated differentiation along with the reduced gene expression of scavenger receptors, CD36 and SR-A, in both PMA- and oxidized low-density lipoprotein (oxLDL)-stimulated THP-1 monocytes. Furthermore, DTF treatment of monocytes and macrophages led to reduction of fluorescent DiI-acLDL and DiI-oxLDL uptake. In conclusion, at least three mechanisms are at work in parallel: DTF reduces LDL oxidation, attenuates monocyte differentiation into macrophage and blunts uptake of modified LDL by macrophage. The effect is different from that of NOB, from which DTF is derived. This study thus significantly enhanced our understanding on how DTF may be beneficial against atherogenesis.

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.

Tanshinone IIA Downregulates Lipogenic Gene Expression and Attenuates Lipid Accumulation through the Modulation of LXRα/SREBP1 Pathway in HepG2 Cells.

Abnormal and excessive accumulation of lipid droplets within hepatic cells is the main feature of steatosis and nonalcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease (MAFLD). Dysregulation of lipogenesis contributes to hepatic steatosis and plays an essential role in the pathological progress of MAFLD. Tanshinone IIA is a bioactive phytochemical isolated from Salvia miltiorrhiza Bunge and exhibits anti-inflammatory, antiatherosclerotic and antihyperlipidemic effects. In this study, we aimed to investigate the lipid-lowering effects of tanshinone IIA on the regulation of lipogenesis, lipid accumulation, and the underlying mechanisms in hepatic cells. We demonstrated that tanshinone IIA can significantly inhibit the gene expression involved in de novo lipogenesis including FASN, ACC1, and SCD1, in HepG2 and Huh 7 cells. Tanshinone IIA could increase phosphorylation of ACC1 protein in HepG2 cells. We further demonstrated that tanshinone IIA also could suppress the fatty-acid-induced lipogenesis and TG accumulation in HepG2 cells. Furthermore, tanshinone IIA markedly downregulated the mRNA and protein expression of SREBP1, an essential transcription factor regulating lipogenesis in hepatic cells. Moreover, we found that tanshinone IIA attenuated liver X receptor α (LXRα)-mediated lipogenic gene expression and lipid droplet accumulation, but did not change the levels of LXRα mRNA or protein in HepG2 cells. The molecular docking data predicted tanshinone IIA binding to the ligand-binding domain of LXRα, which may result in the attenuation of LXRα-induced transcriptional activation. Our findings support the supposition that tanshinone IIA possesses a lipid-modulating effect that suppresses lipogenesis and attenuates lipid accumulation by modulating the LXRα/SREBP1 pathway in hepatic cells. Tanshinone IIA can be potentially used as a supplement or drug for the prevention or treatment of MAFLD.

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.