Sun-Oh Jeong

Roles of heme oxygenase-1 in curcumin-induced growth inhibition in rat smooth muscle cells.

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-α and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non-stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.

Inhibition of TNF-α, IL-1β, and IL-6 productions and NF-κB activation in lipopolysaccharide-activated RAW 264.7 macrophages by catalposide, an iridoid glycoside isolated from Catalpa ovata G. Don (Bignoniaceae)

Abstract Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniaceae), was found to inhibit the productions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and the activation of nuclear factor κB (NF-κB) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Catalposide also inhibited the expressions of TNF-α, IL-1β, and IL-6 genes and the nuclear translocation of p65 subunit of NF-κB in LPS-activated RAW 264.7 cells. Flow cytometric analysis revealed that catalposide suppressed the binding of FITC-conjugated LPS to CD14 on the surface of cells, probably resulting in the inhibitory effects on TNF-α, IL-1β, and IL-6 productions and NF-κB activation. These findings suggest that catalposide could be an attractive candidate for adjunctive therapy in Gram-negative bacterial infections.

Dimethoxycurcumin, a Synthetic Curcumin Analogue, Induces Heme Oxygenase-1 Expression through Nrf2 Activation in RAW264.7 Macrophages.

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] induces heme oxygenase-1 (HO-1) expression via activation of the nuclear factor-erythroid-2-related factor 2 (Nrf2), whereas tetrahydrocurcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione], one of curcumin in vivo metabolites, has no effect on HO-1 expression and Nrf2 activation. The aim of this study was to investigate whether dimethoxycurcumin [1,7-bis(4,3-dimethoxyphenyl)-1,6-heptadiene-3,5-dione], a synthetic curcumin analogue with higher metabolic stability over curcumin, could induce HO-1 expression to the same extent as curcumin in RAW264.7 macrophages. Dimethoxycurcumin and curcumin, but not tetrahydrocurcumin, induced HO-1 expression and Nrf2 nuclear translocation, suggesting that the unsaturated nature of the diarylheptanoid chain of the compounds are crucial for HO-1 expression and Nrf2 activation. Blockage of Nrf2 synthesis by small interfering RNA abolished HO-1 expression by dimethoxycurcumin, indicating that dimethoxycurcumin may induce HO-1 expression via Nrf2 activation. In comparison, dimethoxycurcumin and curcumin had about the same effect on HO-1 expression, suggesting that dimethoxycurcumin retains the HO-1-inducing activity of its parent compound curcumin in RAW264.7 macrophages.

Dimethoxycurcumin, a synthetic curcumin analogue with higher metabolic stability, inhibits NO production, inducible NO synthase expression and NF-κB activation in RAW264.7 macrophages activated with LPS

Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) in activated macrophages is linked to acute and chronic inflammation. Thus, it would be valuable to develop inhibitors of NO and/or iNOS for potential therapeutic use. We investigated whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue with higher metabolic stability over curcumin, could inhibit NO production and iNOS expression in activated macrophages. RAW264.7 macrophages were activated with lipopolysaccharide (LPS) in the absence or presence of DiMC, which contains four methoxy groups at two aromatic rings, curcumin containing two, bis-demethoxycurcumin (BDMC) containing none, or tetrahydrocurcumin (THC) containing two but lacking conjugated double bonds in the central seven-carbon chain. NO production, iNOS expression and NF-kappaB activity were examined. DiMC, curcumin and BDMC inhibited NO production, iNOS expression and NF-kappaB activation, with DiMC being the most effective, followed by curcumin and BDMC. THC failed to inhibit NO production, iNOS expression and NF-kappaB activation. Our results suggest that DiMC inhibits NO production, iNOS expression and NF-kappaB activation in LPS-activated macrophages, which may be due not only to the conjugated double bonds but also the increased number of methoxy groups.

Salidroside from Rhodiola sachalinensis Protects Neuronal PC12 Cells Against Cytotoxicity Induced by Amyloid‐β

Abstract The amyloid β‐peptide (Aβ)‐induced oxidative stress is a well‐established pathway of neuronal cell death in Alzheimers disease (AD). Salidroside, one of the major compounds from the roots of Rhodiola species (Crassulaceae), was investigated in vitro for its cytoprotection against Aβ‐induced toxicity on rat neuronal PC12 cells. Salidroside significantly reduced Aβ‐induced cytotoxicity in a dose‐dependent manner. Salidroside also reduced Aβ‐mediated intracellular accumulation of reactive oxygen species and malondialdehyde (MDA), a product of lipid peroxides, by preventing Aβ‐induced decline of antioxidant enzyme activities. These results suggest that salidroside protects neuronal PC12 cells from Aβ‐induced cytotoxicity via its antioxidant pathway.

Tetrahydroabietic acid, a reduced abietic acid, inhibits the production of inflammatory mediators in RAW264.7 macrophages activated with lipopolysaccharide.

Abietic acid (AA), the main component of the rosin fraction of oleoresin synthesized by conifer species, has been reported to have anti-inflammatory effects. AA is a weak contact allergen; however, compounds resulting from its oxidation by air elicit stronger allergic response. Hydrogenation of the conjugated double bonds of AA, as in tetrahydroabietic acid (THAA), decreases its susceptibility to air oxidation and would thus reduce the allergenicity of AA. The aim of this study was to investigate whether THAA could exert anti-inflammatory effects to the same extent as AA in RAW264.7 macrophages activated with the endotoxin lipopolysaccharide (LPS). THAA and AA inhibited the production of nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively, in LPS-activated RAW264.7 macrophages. They also inhibited the LPS-induced production of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Both THAA and AA prevented the LPS-induced nuclear translocation of the nuclear factor-κB/p65 subunit, suggesting that THAA may inhibit the production of pro-inflammatory mediators through the same mechanism as AA. In comparison, the anti-inflammatory effects of THAA and AA were almost identical, indicating that THAA retains the anti-inflammatory activity of AA at least in LPS-activated RAW264.7 macrophages.

Nardostachys chinensis induces granulocytic differentiation with the suppression of cell growth through p27Kip1 protein-related G0/G1 phase arrest in human promyelocytic leukemic cells

Abstract Context: Nardostachys chinensis Batalin (Valerianaceae) has been used in Korean traditional medicine to elicit stomachic and sedative effects. However, the anti-leukemic activities of N. chinensis have not been well examined. Objective: To investigate the effect of N. chinensis on differentiation and proliferation in the human promyelocytic leukemic HL-60 cells. Materials and methods: The dried roots and stems of N. chiensis are extracted using hot water and then freeze-dried. The yield of extract was 12.82% (w/w). The HL-60 cells were treated with 25–200 μg/ml of N. chinensis for 72 h or 100 μg/ml of N. chinensis for 24–72 h. Results: Nardostachys chinensis significantly inhibited cell viability dose dependently with an IC50 of 100 μg/ml in HL-60 cells. Nardostachys chinensis induced differentiation of the cells as measured by reduction activity of NBT and expression of CD11b but not of CD14 as analyzed by flow cytometry, which indicates a differentiation toward the granulocytic lineage. Nardostachys chinensis also induced growth inhibition through G0/G1 phase arrest in the cell cycle of HL-60 cells. Among the G0/G1 phase in the cell cycle-related protein, the expression of cyclin-dependent kinase (CDK) inhibitor p27Kip1 was increased in N. chinensis-treated HL-60 cells, whereas the expression levels of CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and cyclin A were decreased. Interestingly, N. chinensis markedly enhanced the binding of p27Kip1 with CDK2 and CDK6. Discussion and conclusion: This study demonstrated that N. chinensis is capable of inducing cellular differentiation and growth inhibition through p27Kip1 protein-related G0/G1 phase arrest in HL-60 cells.

In vitro metabolism of the new insecticide flupyrazofos by rat liver microsomes

1. The in vitro metabolism of the new insecticide flupyrazofos was studied using rat liver microsomes. Two metabolites were produced and identified as O,O-diethyl O-(1-phenyl-3-trifluoromethyl-5-pyrazoyl) phosphoric acid ester (flupyrazofos oxon) and 1-phenyl-3-trifluoromethyl-5-hydroxypyrazole (PTMHP) based on UV and mass spectral analysis. 2. Cytochrome P450 oxidatively converted flupyrazofos to flupyrazofos oxon, a major metabolite and phenobarbital-induced microsomes increased this desulphuration by 8-fold. 3. Flupyrazofos oxon was converted to PTMHP with a half-life of 47.8 min by chemical hydrolysis and this conversion also proceeded non-enzymatically under our microsomal incubation conditions.

Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells

Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimers disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases.