Pei-Yi Chen

Curcuminoids Promote Neurite Outgrowth in PC12 Cells through MAPK/ERK- and PKC-Dependent Pathways

Curcuminoids, the predominant polyphenolic compounds in the rhizome of Curcuma longa Linn., consist of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). They exhibit multiple desirable characteristics for a neuroprotective agent including antioxidant, anti-inflammatory, and antiamyloid activities. In this work, we report the first investigation of the neurotrophic action and mechanism of curcuminoids in PC12 cells, which respond to nerve growth factor (NGF) and therefore serve as a model system for primary neuronal cells. The percentages of neurite-bearing cells for those treated with 20 μM curcumin, DMC, and BDMC for 72 h reached 21.6 ± 2.0%, 16.3 ± 2.4%, and 19.9 ± 2.5%, respectively, and were significantly higher than that of the negative control (2.0 ± 0.3%, p < 0.05). In parallel, increased expression of the neuronal differentiation markers, growth-associated protein-43 (GAP-43), and neurofilament-L (NF-L) was found in curcuminoid-treated cells. All three curcuminoids (20 μM) activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase C (PKC) signalings, and inhibition of these kinases with the respective pharmacological inhibitors effectively attenuated curcuminoid-induced neurite outgrowth. Furthermore, our results show that both curcumin and DMC, but not BDMC, induced phosphorylation of cAMP response element-binding protein (CREB) and CRE-reporter gene activity significantly (p < 0.05). These inductions were markedly attenuated by the addition of MEK/ERK or PKC inhibitor; as a consequence, ERK- and PKC-dependent pathways may be involved in curcuminoid-mediated neuritogenesis in PC12 cells. Moreover, activation of CREB coupling with CRE-dependent gene transcription may play a vital role for curcumin- or DMC-induced PC12 differentiation.

Curcumin enhances cell-surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene expression in HepG2 cells.

SCOPE Curcumin has been demonstrated as having numerous desirable characteristics, such as antioxidant, anti-inflammatory, and antiatherogenic activities. We report the hypocholesterolemic effect and molecular mechanism of curcumin. METHODS AND RESULTS We found that curcumin enhanced LDL receptor (LDLR) level on the cell surface, as well as LDLR activity; however, LDLR transcription and mRNA stability were not affected. Furthermore, we found that proprotein convertase subtilisin/kexin type 9 (PCSK9) gene was downregulated at the transcriptional level by curcumin, leading to an increase in LDL uptake in HepG2 cells. The curcumin-responsive element of the PCSK9 promoter, a binding site for hepatocyte nuclear factor 1α (HNF-1α), was also identified. We demonstrated that curcumin reduced the nuclear abundance of hepatocyte nuclear factor 1α, resulting in its attenuated interaction with the PCSK9 promoter and leading to a downregulation of PCSK9 expression. Finally, we showed that curcumin decreased the statin-induced PCSK9 expression and potentially synergized with statin administration. CONCLUSION Current results indicate that curcumin suppression of PCSK9 expression is associated with increases in cell-surface LDLR and LDLR activity in hepatic cells and it acts in a molecular mechanism that is distinct from the statins. Curcumin exhibits hypolipidemic activity and may serve as a useful supplement to statin treatment for hypercholesterolemia.

Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.

Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells

Luteolin (3′,4′,5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.

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.

Neurotrophic Action of 5-Hydroxylated Polymethoxyflavones: 5-Demethylnobiletin and Gardenin A Stimulate Neuritogenesis in PC12 Cells

Polymethoxyflavones (PMFs) exhibit a broad spectrum of biological properties, including anticancer, antiatherogenic, and neuroprotective effects. The aim of this study is to investigate the neurotrophic effects of 5-demethylnobiletin, a hydroxylated PMF found in citrus plants, and gardenin A, a synthetic PMF analogue, on neurite outgrowth and neuronal differentiation in PC12 cells. The results of this study showed that 5-demethylnobiletin and gardenin A (10-20 μM) potently induce neurite outgrowth in PC12 cells, accompanied by the expression of neuronal differentiation and synapse formation marker proteins, growth-associated protein-43 (GAP-43), and synaptophysin. We observed that the addition of K252a, a TrKA antagonist, significantly inhibited NGF-induced neurite outgrowth in PC12 cells, but 5-demethylnobiletin- or gardenin A-induced neurite outgrowth was not affected. Treatment with 5-demethylnobiletin and gardenin A markedly induced the phosphorylation of both cyclic AMP response element-binding protein (CREB) and CRE-mediated transcription, which was suppressed through the administration of the inhibitor 2-naphthol AS-E phosphate (KG-501) or using CREB siRNA. Furthermore, our results showed that MAPK/ERK kinase 1/2 (MEK1/2), protein kinase A (PKA), and protein kinase C (PKC) inhibitors blocked the CRE transcription activity and neurite outgrowth induced through 5-demethylnobiletin or gardenin A. Consistently, increased ERK phosphorylation and PKA and PKC activities were observed in PC12 cells treated with 5-demethylnobiletin or gardenin A. These results reveal for the first time that 5-demethylnobiletin and gardenin A promote neuritogenesis through the activation of MAPK/ERK-, PKC-, and PKA-dependent, but not TrkA-dependent, CREB signaling pathways in PC12 cells.

Curcuminoids Modulate the PKCδ/NADPH Oxidase/Reactive Oxygen Species Signaling Pathway and Suppress Matrix Invasion during Monocyte-Macrophage Differentiation.

Monocyte recruitment and invasion play critical roles in the initiation and progression of atherosclerosis. The reduction in monocyte adhesion and infiltration is thought to exert antiatherosclerotic effects. Curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are the major active components of curcuminoids and exhibit several biological activities, including anti-inflammatory, anticarcinogenic, and hypocholesterolemic activities. The aim of this study was to investigate the antiatherogenic effects and mechanisms of curcuminoids during monocyte to macrophage differentiation. The results showed that curcumin, DMC, and BDMC (20 μM) suppressed matrix invasion from 100.0 ± 5.0% to 24.8 ± 1.4%, 26.6 ± 2.9%, and 33.7 ± 1.7%, respectively, during PMA-induced THP-1 differentiation. We found that curcuminoids significantly reduced PMA-induced CD11b and MMP-9 expression by THP-1 cells. Production of reactive oxygen species (ROS) induced by PMA (126.7 ± 2.1%) was markedly attenuated by curcumin, DMC, and BDMC to 99.5 ± 7.8%, 87.8 ± 8.2%, and 89.8 ± 7.6%, respectively, resulting in the down-regulation of CD11b and MMP-9 expression. We demonstrated that curcuminoids inhibited NADPH oxidase through the down-regulation of NOX2 expression and the reduction of p47phox membrane translocation. Moreover, we found involvement of PKCδ in the PMA-induced NOX2, CD11b, and MMP-9 mRNA expression. Curcumin, DMC, and BDMC decreased the active form of PKCδ protein stimulated by PMA in THP-1 cells. Overall, our results reveal that curcuminoids suppress matrix invasion through the inhibition of the PKCδ/NADPH oxidase/ROS signaling pathway during monocyte-macrophage differentiation.

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.

Prenatal diagnosis of the maternal derivative chromosome der(15)t(Y;15)(q12;p13) in a dizygotic twin pregnancy

Sex chromosome translocations are unique and must be considered separately from translocations between autosomes. Here, we describe the first prenatal case of one twin fetus with an unbalanced translocation between chromosome Y and chromosome 15, presenting a 46,XY,der(15)t(Y;15) karyotype. The other twin had a normal 46,XY karyotype. Cytogenetic analysis of the parental chromosomes revealed that the father had a normal 46,XY karyotype, whereas the mother exhibited a 46,XX,der(15) t(Y;15) karyotype. Thus, the proband inherited this translocation from the mother. Fluorescence in situ hybridization analyses demonstrated that the breakpoint on chromosome Y involved a heterochromatin region (Yq12), while that on chromosome 15 involved a p-arm region (15p13). At 37 gestational weeks, healthy twins were delivered vaginally. We conclude that accurate identification of der(15) chromosomal content can facilitate not only prenatal diagnosis of a chromosomal aberration in one twin, but also prediction of the fetal phenotype.

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.