Tran Thi Hien

Metformin inhibits P-glycoprotein expression via the NF-κB pathway and CRE transcriptional activity through AMPK activation

BACKGROUND AND PURPOSE The expression of P‐glycoprotein (P‐gp), encoded by the multidrug resistance 1 (MDR1) gene, is associated with the emergence of the MDR phenotype in cancer cells. We investigated whether metformin (1,1‐dimethylbiguanide hydrochloride) down‐regulates MDR1 expression in MCF‐7/adriamycin (MCF‐7/adr) cells.

Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

Molecular mechanism of suppression of MDR1 by puerarin from Pueraria lobata via NF‐κB pathway and cAMP‐responsive element transcriptional activity‐dependent up‐regulation of AMP‐activated protein kinase in breast cancer MCF‐7/adr cells

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy and its inhibition is an effective way to reverse cancer drug resistance. In the present study, we investigated that puerarin, a natural isoflavonoid from Pueraria lobata, down-regulated MDR1 expression in MCF-7/adriamycin (MCF-7/adr), a human breast MDR cancer cell line. Puerarin treatment significantly inhibited MDR1 expression, MDR1 mRNA and MDR1 promoter activity in MCF-7/adr cells. The suppression of MDR1 was accompanied by partial recovery of intracellular drug accumulation, leading to increased toxicity of adriamycin and fluorescence of rhodamine 123, indicating that puerarin reversed the MDR phenotype by inhibiting the drug efflux function of MDR1. Moreover, nuclear factor kappa-B activity and IkappaB degradation were inhibited by puerarin. Puerarin stimulated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase and glycogen synthase kinase-3beta phosphorylation, but puerarin decreased cAMP-responsive element-binding protein phosphorylation. The puerarin-induced suppression of MDR1 expression was reduced by AMPK inhibitor (compound C). Furthermore, both MDR1 protein expression and the transcriptional activity of cAMP-responsive element (CRE) were inhibited by puerarin and protein kinase A/CRE inhibitor (H89). Taken together, our results suggested that puerarin down-regulated MDR1 expression via nuclear factor kappa-B and CRE transcriptional activity-dependent up-regulation of AMPK in MCF-7/adr cells.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase.

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N(G)-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.

Pin1 induction in the fibrotic liver and its roles in TGF-β1 expression and Smad2/3 phosphorylation

BACKGROUND & AIMSnTherapeutic management of liver fibrosis remains an unsolved clinical problem. Hepatic accumulation of extracellular matrix, mainly collagen, is mediated by the production of transforming growth factor-β1 (TGF-β1) in stellate cells. Pin1, a peptidyl-prolyl isomerase, plays an important pathophysiological role in several diseases, including neurodegeneration and cancer. Herein, we determined whether Pin1 regulates liver fibrogenesis and examined its mechanism of action by focusing on TGF-β1 signalling and hepatic stellate cell (HSC) activation.nnnMETHODSnPin1 expression was assessed by immunohistochemistry, Western blot or real-time-polymerase chain reaction (RT-PCR) analyses of human and mouse fibrotic liver samples. The role of Pin1 during HSC activation was estimated using Pin1-null mouse embryonic fibroblast (MEF) cells and Pin1-overexpressing LX-2 human hepatic stellate cells.nnnRESULTSnPin1 expression was elevated in human and mouse fibrotic liver tissues, and Pin1 inhibition improved dimethylnitrosamine (DMN)-induced liver fibrosis in mice. Pin1 inhibition reduced the mRNA or protein expression of TGF-β1 and α-smooth muscle actin (α-SMA) by DMN treatment. Pin1 knockdown suppressed TGFβ1 gene expression in both LX-2 and MEF cells. Pin1-mediated TGFβ1 gene transcription was controlled by extracellular signal-regulated kinase (ERK)- and phosphoinositide 3-kinase/Akt-mediated activator protein-1 (AP-1) activation. Moreover, TGFβ1-stimulated Smad2/3 phosphorylation and plasminogen activator inhibitor-1 expression were inhibited by Pin1 knockdown.nnnCONCLUSIONSnPin1 induction during liver fibrosis is involved in hepatic stellate cell activation, TGFβ1 expression, and TGFβ1-mediated fibrogenesis signalling.

Role of Pin1 in neointima formation: down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr-Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3weeks (1mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

Ultraviolet A (UVA) radiation (λ = 320-400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, we demonstrated that Pin1 expression was enhanced by low energy UVA (300-900 mJ/cm(2)) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.

Nectandrin B suppresses the expression of adhesion molecules in endothelial cells: Role of AMP-activated protein kinase activation

We have previously shown that nectandrin B, a potent natural activator of AMP-activated protein kinase (AMPK) results in endothelium-dependent relaxation via endothelial nitric oxide synthase phosphorylation. This study examined the effects of nectandrin B on monocyte adhesion and on the expression of adhesion molecules in endothelial cells, an initial event in atherogenesis. Nectandrin B inhibited tumor necrosis factor-α (TNFα)-induced monocytoid THP-1 cell adhesion to ECV 304 human endothelial cells. This lignan also suppressed TNFα-induced protein and mRNA expression of two cell adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). In addition, expression of cyclooxygenase-2 and inducible nitric oxide synthase were diminished by nectandrin B treatment. Reporter gene and immunoblot analyses revealed that transcription factor activities of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and cyclic AMP response element binding protein (CREB) were inhibited by nectandrin B. Moreover, nectandrin B activated AMP-activated protein kinase (AMPK) in ECV 304 cells. Transfection of a dominant-negative mutant form of AMPK (DN-AMPK) partially reversed inhibitory effects of nectandrin B on the expression of VCAM-1 and ICAM-1, and on the transcriptional activity of CREB.

Molecular mechanism of endothelial nitric-oxide synthase activation by Platycodon grandiflorum root-derived saponins.

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) has antithrombotic and antiatherosclerotic properties in the vasculature. Previously, we demonstrated that saponins derived from the roots of Platycodon grandiflorum (CKS) inhibited the tumor necrosis factor-alpha-induced expression of adhesion molecules in human endothelial cells. In this study, we found that CKS increased eNOS phosphorylation and NO production in human endothelial cells. Treatment with CKS increased the phosphorylation of Akt, p38/MAPK, AMP-activated protein kinase (AMPK), and calmodulin-dependent protein kinase II (CaMK II) leading to increased NO production in human endothelial cells. Moreover, inhibitors of Akt (LY294002), p38/MAPK (SB203580), AMPK (compound C), and CaMK II (W7) failed to suppress CKS-induced eNOS phosphorylation. In addition, CKS-induced eNOS phosphorylation was inhibited by the overexpression of a dominant-negative mutant form of AMPK (DN-AMPK). Taken together, these results indicate that CKS stimulates eNOS phosphorylation and NO production via the activation of PI3K/Akt, p38/MAPK, AMPK, and CaMK II.

Evaluation of antioxidant defense systems in H4IIE cells infected with a retroviral vector

Retroviral gene transfer technology is frequently used to establish stable transgenic cell lines. However, no studies to date have evaluated antioxidant defense systems in cells infected with retroviral particles. In the present study, we examined the effects of retroviral infection on antioxidant defense systems using H4IIE cells infected with a retrovirus that overexpresses green fluorescent protein (retro-H4IIE cells). Total oxyradical scavenging capacity and glutathione (GSH), malondialdehyde, and peroxide levels were not significantly altered in retro-H4IIE cells; however, retro-H4IIE cells showed a higher resistance against cytotoxicity, GSH depletion, and malondialdehyde elevation under H(2)O(2)-induced oxidative stress conditions. Immunoblot analysis showed that alpha-class GSH S-transferase (GST) was increased 2.5-fold in retro-H4IIE cells as compared with H4IIE cells; however, catalase, GSH peroxidase-1, peroxiredoxin-1, and thioredoxin-1 remained unaltered or slightly decreased. l-Buthionine-(S,R)-sulfoximine, a GSH synthesis inhibitor, and 1-chloro-2,4-dinitrobenzene, a GST substrate and competitive inhibitor, decreased the difference in H(2)O(2) responses between the two cell types. These results support the hypothesis that the resistance of retro-H4IIE cells to H(2)O(2) can be attributed to an increase in alpha-class GST expression, as levels of GSH and GSH peroxidase-1 were not altered. The present study suggests that antioxidant enzyme expression may change during the establishment of stable transformed cell lines using retroviral techniques.