Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum.

Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl)-1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O2·â») production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O2·â» production. The peak cytosolic calcium concentration ([Ca²âº](i)) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca²âº](i) was significantly shortened. In a calcium-free solution, changes in [Ca²âº](i) caused by the addition of extracellular Ca²âº were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca²âº](i) changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE.

Thrombin induces inducible nitric oxide synthase expression via the MAPK, MSK1, and NF-κB signaling pathways in alveolar macrophages.

In this study, we investigated the roles of mitogen activated protein kinase (MAPK), mitogen stress-activated protein kinase 1 (MSK1), and nuclear factor-κB (NF-κB) signaling pathways in thrombin-induced inducible nitric oxide synthase (iNOS) expression in alveolar macrophages (NR8383). Treatment of NR8383 cells with thrombin caused an increase in iNOS expression in a concentration- and time-dependent manner. Treatment of NR8383 cells with SB203580 (4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole, a p38 MAPK inhibitor), PD98059 (2'-amino-3'-methoxyflavone, a MAPK kinase (MEK) inhibitor), and SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one, a JNK inhibitor) all inhibited thrombin-induced iNOS expression. Stimulation of cells with thrombin caused an increase in p38 MAPK, ERK, and JNK phosphorylation. Treatment of cells with Ro 31-8220 (an MSK1 inhibitor) and MSK1 small interfering RNA (MSK1 siRNA) both inhibited thrombin-induced iNOS expression. Thrombin caused time-dependent activation of MSK1 Ser531 phosphorylation, which was inhibited by SB203580 and PD98059, but not by SP600125. Treatment of cells with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) inhibited thrombin-induced iNOS expression in a concentration-dependent manner. Treatment of NR8383 cells with thrombin induced κB-luciferase activity and p65 Ser276 phosphorylation. Thrombin-induced increases in p65 Ser276 phosphorylation and κB-luciferase activity were inhibited by SB203580, PD98059, Ro 31-8220, and MSK1 siRNA. Taken together, these results suggest that the signaling pathways of MAPK, MSK1, and NF-κB play important roles in thrombin-induced iNOS expression in alveolar macrophages.

Involvement of phosphatidylcholine-phospholipase C and protein kinase C in peptidoglycan-induced nuclear factor-kappaB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages.

In this study, we examined the role of phosphatidylcholine-phospholipase C (PC-PLC) and protein kinase C (PKC) in peptidoglycan (PGN)-induced nuclear factor-kappaB (NF-kappaB) activation and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a PC-PLC inhibitor (D609) and by PKC inhibitors (Go 6976 and Ro 31-8220), but not by a phosphatidylinositol-PLC (PI-PLC) inhibitor (U-73122). PGN caused an increase in PKC activity, and this effect was inhibited by D609, Go 6976, and Ro 31-8220, but not by U-73122. Furthermore, the PGN-mediated increases in kappaB-luciferase activity were also inhibited by D609 and Ro 31-8220. Our data demonstrate that PGN activates PC-PLC which induces PKC activation; this in turn initiates NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

Involvement of hypoxia-inducing factor-1alpha-dependent plasminogen activator inhibitor-1 up-regulation in Cyr61/CCN1-induced gastric cancer cell invasion.

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.