Jeong-Hyung Lee

Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells

Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor-beta superfamily, which is overexpressed in a variety of human cancers, including breast and gastric cancer. The function of MIC-1 in cancer remains controversial and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of ErbB2 in SK-BR-3 breast and SNU-216 gastric cancer cells. MIC-1 induced a significant phosphorylation of Akt and ERK-1/2, and also effected an increase in the levels of tyrosine phosphorylation of ErbB1, ErbB2 and ErbB3 in SK-BR-3 and SNU-216 cells. The treatment of these cells with AG825 and AG1478, inhibitors specific for ErbB2 tyrosine kinase, resulted in the complete abolition of MIC-1-induced Akt and ERK-1/2 phosphorylation. Furthermore, the small-interfering RNA-mediated downregulation of ErbB2 significantly reduced not only the phosphorylation of Akt and ERK-1/2 but also the invasiveness of the cells induced by MIC-1. Our results show that ErbB2 activation performs a crucial function in MIC-1-induced signaling pathways. Further investigations revealed that MIC-1 induced the expression of the hypoxia inducible factor-1alpha protein and the expression of its target genes, including vascular endothelial growth factor, via the activation of the mammalian target of rapamycin (mTOR) signaling pathway. Stimulation of SK-BR-3 with MIC-1 profoundly induces the phosphorylation of mTOR and its downstream substrates, including p70S6K and 4E-BP1. Collectively, these results show that MIC-1 may participate in the malignant progression of certain human cancer cells that overexpress ErbB2 through the transactivation of ErbB2 tyrosine kinase.

The anti-inflammatory effect of tussilagone, from Tussilago farfara, is mediated by the induction of heme oxygenase-1 in murine macrophages.

Tussilagone (TSL), isolated from the flower of buds of Tussilago farfara (Compositae), is a sesquiterpenoid that is known to exert a variety of pharmacological activities. In the present study, we demonstrated that TSL exerts anti-inflammatory activities in murine macrophages by inducing heme oxygenase-1 (HO-1) expression. Treatment of RAW264.7 cells with TSL-induced HO-1 protein expression in a dose- and time-dependent manner without the induction of HO-1 mRNA expression. TSL-mediated HO-1 protein induction was not inhibited by treatment with actinomycin D, a transcriptional inhibitor, but by cycloheximide, a translational inhibitor. Moreover, mitogen-activated protein kinases (MAPKs) inhibitors such as SB203580, SP600125, and U0126 did not block TSL-mediated HO-1 protein expression, suggesting that the TSL-mediated HO induction may be regulated at the translational level. Consistent with the notion that HO-1 has anti-inflammatory properties, TSL inhibited the production of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin E2 (PGE2) as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and murine peritoneal macrophages. Inhibition of HO-1 activity by treatment with zinc protoporphyrin IX (ZnPP), a specific HO-1 inhibitor, abrogated the inhibitory effects of TSL on the production of NO and PGE2 in LPS-stimulated RAW264.7 cells. Taken together, TSL may be an effective HO-1 inducer that has anti-inflammatory effects, and a valuable compound for modulating inflammatory conditions.

Tangeretin, a citrus flavonoid, inhibits PGDF-BB-induced proliferation and migration of aortic smooth muscle cells by blocking AKT activation

Tangeretin, a natural polymethoxylated flavone concentrated in the peel of citrus fruits, is known to have antiproliferative, antiinvasive, antimetastatic and antioxidant activities. However, the effect of tangeretin on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of tangeretin on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Tangeretin significantly inhibited proliferation, DNA synthesis and migration of PDGF-BB-stimulated RASMCs without inducing cell death. Treatment with tangeretin-induced cell-cycle arrest in the G₀/G₁ phase was associated with down-regulation of cyclin D1 and cyclin E in addition to up-regulation of p27(kip1). We also showed that tangeretin inhibited PDGF-BB-induced phosphorylation of AKT, while it had no effect on the phosphorylation of phospholipase Cγ (PLCγ), PDGF receptor β-chain (PDGF-Rβ) and extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs). An in vitro kinase assay revealed that tangeretin inhibited AKT activity in a dose-dependent manner. Moreover, treatment of LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, had similar effects than that of tangeretin on the expression of p27(kip1) and cyclin D1, as well as cell migration in PDFG-BB-stimulated RASMCs. Taken together, these findings suggest that tangeretin could suppress PDGF-BB-induced proliferation and migration of RASMCs through the suppression of PI3K/AKT signaling pathway, and may be a potential candidate for preventing or treating vascular diseases, such as atherosclerosis and restenosis.

Eupatolide inhibits lipopolysaccharide-induced COX-2 and iNOS expression in RAW264.7 cells by inducing proteasomal degradation of TRAF6

Inula britannica is a traditional medicinal plant used to treat bronchitis, digestive disorders, and inflammation in Eastern Asia. Here, we identified eupatolide, a sesquiterpene lactone from I. britannica, as an inhibitor of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Eupatolide inhibited the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) as well as iNOS and COX-2 protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Eupatolide dose-dependently decreased the mRNA levels and the promoter activities of COX-2 and iNOS in LPS-stimulated RAW264.7 cells. Moreover, eupatolide significantly suppressed the LPS-induced expression of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) reporter genes. Pretreatment of eupatolide inhibited LPS-induced phosphorylation and degradation of I kappaB alpha, and phosphorylation of RelA/p65 on Ser-536 as well as the activation of mitogen-activated protein kinases (MAPKs) and Akt in LPS-stimulated RAW264.7 cells. Eupatolide induced proteasomal degradation of tumor necrosis factor receptor-associated factor-6 (TRAF6), and subsequently inhibited LPS-induced TRAF6 polyubiquitination. These results suggest that eupatolide blocks LPS-induced COX-2 and iNOS expression at the transcriptional level through inhibiting the signaling pathways such as NF-kappaB and MAPKs via proteasomal degradation of TRAF6. Taken together, eupatolide may be a novel anti-inflammatory agent that induces proteasomal degradation of TRAF6, and a valuable compound for modulating inflammatory conditions.

Polychlorinated compounds with PPAR-γ agonistic effect from the medicinal fungus Phellinus ribis

During the search for natural substances with PPAR-gamma agonistic effect, unique polychlorinated compounds named chlorophellins A-C have been isolated together with the known compound, drosophilin A, from the methanolic extract of the fruiting body of the fungus Phellinus ribis. Their structures were assigned on the basis of NMR and mass spectrometric analyses. Chlorophellin C of compounds tested exhibited the most potent PPAR-gamma agonistic effect and was comparable to rosiglitazone, a well-known PPAR-gamma agonist that has been used for the therapy of type 2 diabetes.

Eupatolide inhibits PDGF-induced proliferation and migration of aortic smooth muscle cells through ROS-dependent heme oxygenase-1 induction.

The abnormal proliferation and migration of vascular smooth muscle cell (VSMC) contributes importantly to the pathogenesis of atherosclerosis and restenosis. Here, we investigated the effects of eupatolide (EuTL), a sesquiterpene lactone isolated from the medicinal plant Inula britannica, on platelet‐derived growth factor (PDGF)‐induced proliferation and migration of primary rat aortic smooth muscle cells (RASMCs), as well as its underlying mechanisms. EuTL remarkably inhibited PDGF‐induced proliferation and migration of RASMCs. Treatment of RASMCs with EuTL induced both protein and mRNA expression of heme oxygenase‐1 (HO‐1). SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress EuTL‐induced HO‐1 expression; however, N‐acetylcysteine (NAC, an antioxidant) blocked EuTL‐induced HO‐1 expression. Moreover, treatment of RASMCs with EuTL increased reactive oxygen species (ROS) accumulation and nuclear translocation of nuclear factor‐E2‐related factor 2 (Nrf2); however, this translocation was also inhibited by NAC. NAC or inhibition of HO‐1 significantly attenuated the inhibitory effects of EuTL on PDGF‐induced proliferation and migration of RASMCs. Taken together, these findings suggest that EuTL could suppress PDGF‐induced proliferation and migration of VSMCs through HO‐1 induction via ROS‐Nrf2 pathway and may be a potential HO‐1 inducer for preventing or treating vascular diseases. Copyright

Malabaricone C suppresses lipopolysaccharide-induced inflammatory responses via inhibiting ROS-mediated Akt/IKK/NF-κB signaling in murine macrophages

Malabaricone C (MLB-C), isolated from nutmeg, is a phenolic diarylnonanoid that is known to exert a variety of pharmacological activities. In the present study, we investigated the molecular actions of MLB-C against lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells and murine peritoneal macrophages. MLB-C inhibited the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-6 (IL-6), and interferon-γ (INF-γ) in a dose-dependent manner. Consistent with NO and PGE(2) inhibition, MLB-C suppressed LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression as well as the promoter activities of COX-2 and iNOS. MLB-C pretreatment prevented LPS-induced nuclear factor-kappa B (NF-κB) activation through the inhibition of phosphorylation of IκB kinase (IKK), phosphorylation and degradation of IκBα, and nuclear translocation of NF-κB. In addition, MLB-C blocked LPS-induced serine 536 phosphorylation and transcriptional activity of RelA/p65 subunit of NF-κB. Further study demonstrated that MLB-C inhibited LPS-induced Akt phosphorylation, which is an upstream activator of NF-κB, by reducing reactive oxygen species (ROS) accumulation, without affecting phosphorylation of mitogen-activated protein kinases (MAPKs). These findings indicate that MLB-C exerts an anti-inflammatory effect through the inhibition of NF-κB activation by inhibiting interconnected ROS/Akt/IKK/NF-κB signaling pathways.

Sappanone A exhibits anti-inflammatory effects via modulation of Nrf2 and NF-κB.

UNLABELLED Homoisoflavonoids constitute a small class of natural products. In the present study, we investigated the anti-inflammatory effect of sappanone A (SPNA), a homoisoflavanone that is isolated from the heartwood of Caesalpinia sappan (Leguminosae), in murine macrophages. SPNA inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6) as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Moreover, SPNA protected C57BL/6 mice from LPS-induced mortality. Treatment of RAW264.7 cells with SPNA induced heme oxygenase (HO)-1 protein and mRNA expression and increased nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (NQO1). Knockdown of Nrf2 by siRNA blocked SPNA-mediated HO-1 induction. SB203580, p38 mitogen-activated protein kinase (MAPK) inhibitor, blocked SPNA-induced HO-1 expression and nuclear translocation of Nrf2, suggesting that SPNA induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of SPNA in LPS-stimulated RAW264.7 cells. Moreover, SPNA suppressed LPS-induced nuclear factor κB (NF-κB) activation via inhibiting Ser 536 phosphorylation and transcriptional activity of RelA/p65 subunit of NF-κB. Taken together, these findings suggest that SPNA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways, and may be a valuable compound to prevent or treat inflammatory diseases.

Anti-inflammatory and heme oxygenase-1 inducing activities of lanostane triterpenes isolated from mushroom Ganoderma lucidum in RAW264.7 cells

Ganoderma lucidum is a popular medicinal mushroom used in traditional medicine for preventing or treating a variety of diseases. In the present study, we investigated the anti-inflammatory and heme oxygenase (HO)-1 inducing effects of 12 lanostane triterpenes from G. lucidum in RAW264.7 cells. Of these, seven triterpenes, butyl lucidenateE2, butyl lucidenateD2 (GT-2), butyl lucidenate P, butyl lucidenateQ, Ganoderiol F, methyl ganodenate J and butyl lucidenate N induced HO-1 expression and suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Inhibiting HO-1 activity abrogated the inhibitory effects of these triterpenes on the production of NO in LPS-stimulated RAW264.7 cells, suggesting the involvement of HO-1 in the anti-inflammatory effects of these triterpenes. We further studied the anti-inflammatory and HO-1 inducing effects of GT-2. Mitogen-activated protein kinase inhibitors or N-acetylcysteine, an antioxidant, did not suppress GT-2-mediated HO-1 induction; however, LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked GT-2-induced HO-1 mRNA and protein expression. GT-2 increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and knockdown of Nrf2 by small interfering RNA blocked GT-2-mediated HO-1 induction, suggesting that GT-2 induced HO-1 expression via the PI3K/AKT-Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, GT-2 inhibited the production of tumor necrosis factor-α and interleukin-6, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression. These findings suggest that HO-1 inducing activities of these lanostane triterpenes may be important in the understanding of a novel mechanism for the anti-inflammatory activity of G. lucidum.

EFFECTS OF CALCIUM CHANNEL BLOCKERS ON THE SPERMATOGENESIS AND GENE EXPRESSION IN PERIPUBERTAL MOUSE TESTIS

Treatment of Ca2+ channel blockers (CCB) to relieve hypertension causes reversible male infertility, suggesting deregulation of Ca2+ homeostasis in testis is closely related with male infertility. To investigate the possible toxicity of therapeutic application of CCB in childhood, the effect of nifedipine and ethosuximide, an L-type and T-type CCB, respectively, on the spermatogenesis and testicular gene expression was examined. Following the intraperitoneal injection of either drug for 7 days to 18 days on old mice, the paired testes weights were significantly lower in mice treated with nifedipine (≥ 10 mg/kg/day) or ethosuximide (100 mg/kg/day) than vehicle controls. In mice given high drug dosing (100 mg/kg), seminiferous tubules showed immaturity with spermatogenic arrest at elongating spermatid stage and poorly developed lumen. Unexpectedly, the expression of activator isoform of transcription factor cAMP-responsive element modulator (CREM) mRNA increased together with transition protein 2 and protamine 2 mRNA in drug-treated mice testes, suggesting that CCB may deregulate expression of activator isoform of CREM in male germ cells and that spermatogenic defect following CCB treatment may attribute to ectopic expression of CREM-dependent gene battery in testis. Therapeutic application of CCB in childhood should be cautious because of their potential to cause spermatogenic defect and altered gene expression in testis.