Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway.

Connective tissue growth factor (CTGF) plays an important role in lung fibrosis. In this study, we investigated the role of Rac1, mixed-lineage kinase 3 (MLK3), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CTGF-induced collagen I expression in human lung fibroblasts. CTGF caused concentration- and time-dependent increases in collagen I expression. CTGF-induced collagen I expression was inhibited by the dominant negative mutant (DN) of Rac1 (RacN17), MLK3DN, MLK3 inhibitor (K252a), JNK1DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). Treatment of cells with CTGF caused activation of Rac1, MLK3, JNK, and AP-1. The CTGF-induced increase in MLK3 phosphorylation was inhibited by RacN17. Treatment with RacN17 and the MLK3DN inhibited CTGF-induced JNK phosphorylation. CTGF caused increases in c-Jun phosphorylation and the recruitment of c-Jun and c-Fos to the collagen I promoter. Furthermore, stimulation of cells with the CTGF resulted in increases in AP-1-luciferase activity; this effect was inhibited by Rac1N17, MLK3DN, JNK1DN, and JNK2DN. Moreover, CTGF-induced α-smooth muscle actin (α-SMA) expression was inhibited by the procollagen I small interfering RNA (siRNA). These results suggest for the first time that CTGF acting through Rac1 activates the MLK3/JNK signaling pathway, which in turn initiates AP-1 activation and recruitment of c-Jun and c-Fos to the collagen I promoter and ultimately induces collagen I expression in human lung fibroblasts.

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase.

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.

Baicalein induction of hydroxyl radical formation via 12-lipoxygenase in human platelets: an ESR study.

The pro-oxidant activities of baicalein, morin, myricetin, quercetin, and rutin were examined in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs), human THP-1 cells, and fibroblast cells. Electron spin resonance (ESR) results showed that only baicalein generated hydroxyl radicals in a resting human platelet suspension, whereas the other flavonoids showed no effects on any of the resting cell systems. A low concentration of arachidonic acid (AA) increased the intensity of hydroxyl radicals, but a high concentration inhibited it. Collagen and thrombin, platelet aggregatory agents that can cause the release of AA by platelets, enhanced baicalein-induced hydroxyl radical formation, whereas ADP and U44619 showed no significant effects. Quinacrine and 5,8,11,14-eicosatetraenoic trifluoromethyl ketone, both PLA2 inhibitors, significantly attenuated baicalein-induced hydroxyl radical formation. These results suggest that baicalein-induced hydroxyl radical formation is associated with AA metabolite enzymes in human platelets. The formation of hydroxyl radicals was significantly inhibited by lipoxygenase inhibitors including nordihydroguaiaretic acid, (-)-epicatechin, (-)-epicatechin gallate, and hinokitiol, but was not affected by desferroxamine or the heme protein inhibitors KCN and NaN3. On the other hand, semiquinone free radicals were generated when baicalein was incubated with horseradish peroxidase/H2O2 or platelets/AA. The semiquinone radicals formed in the platelets/AA system could be extensively inhibited by desferroxamine, diethylenetriaminepentaacetic acid, KCN, and NaN3, indicating that prostaglandin H synthase (PGHS)-peroxidase may be involved. The results of this study led to the proposal that baicalein induces hydroxyl radical formation via 12-lipoxygenase and induces semiquinone radical formation via PGHS-peroxidase in human platelets.

Inhibitory mechanisms of low concentrations of oxidized low-density lipoprotein on platelet aggregation.

The intracellular mechanisms underlying oxidized low-density lipoprotein (oxLDL)-signaling pathways in platelets are not yet completely understood. Therefore, the aim of this study was to further examine the effects of oxLDL in prevention of platelet aggregation. In this study, oxLDL concentration-dependently (40-120 microg/ml) inhibited platelet aggregation in human platelet-rich plasma stimulated by agonists. Moreover, oxLDL (40 and 80 microg/ml) markedly decreased the fluorescence intensity of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (150 nM). This phosphorylation was markedly inhibited by oxLDL (40 and 80 microg/ml) in phosphorus-32-labeled platelets. In addition, oxLDL (40 and 80 microg/ml) markedly increased levels of cyclic AMP and cyclic AMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. The thrombin-evoked increase in pHi was inhibited in the presence of oxLDL (40 and 80 microg/ml). These results indicate that the antiplatelet activity of oxLDL may involve the following pathways. (1) oxLDL may initially induce conformational changes in platelet membranes, leading to inhibition of the activation of protein kinase C, followed by inhibition of P47 protein phosphorylation, and intracellular Ca(2+) mobilization. (2) oxLDL also activated formation of cyclic AMP and cyclic AMP-induced VASP Ser(157) phosphorylation, resulting in inhibition of the Na(+)/H(+)exchanger; this leads to reduced intracellular Ca(2+) mobilization, and ultimately to inhibition of platelet aggregation. This study further provides new insights concerning the effects of low concentrations of oxLDL on platelet aggregation.

ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets.

Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and alpha-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.

Induction of differentiation in rat C6 glioma cells with Saikosaponins.

The effects of saikosaponins (a, b(1), b(2), c, d), isolated from Bupleurum Radix, on the induction of differentiation in rat C6 glioma cells were studied. Saikosaponins a and d were shown to inhibit cell proliferation and alter cell morphology. In addition to cytostasis, the enzymatic activities of glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) were also noticeably increased after treatment with saikosaponin a. Nevertheless, saikosaponin d only showed an increase of GS activity, no significant changes in CNP activity were found. These results suggest that saikosaponin a can induce the differentiation of C6 glioma cells into astrocytes and/or oligodendrocytes, but saikosaponin d can only induce the differentiation of C6 glioma cells into astrocytes.