Solanum nigrum produces nitric oxide via nuclear factor-kappaB activation in mouse peritoneal macrophages.

Nitric oxide (NO) is an antitumour molecule produced in activated macrophages and Solanum nigrum is a plant used in oriental medicine to treat tumours. In this study using mouse peritoneal macrophages, we have examined the mechanism by which Solanum nigrum regulates NO production. When Solanum nigrum was used in combination with 20 U/ml of recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. The increase in NO synthesis was reflected as an increased amount of inducible NO synthase (iNOS) protein. The production of NO from rIFN-gamma plus Solanum nigrum-stimulated peritoneal macrophages was decreased by treatment with N-monomethyl-L-arginine or N-tosyl-Phe chloromethyl ketone, an iNOS inhibitor. Additionally, the increased production of NO from rIFN-gamma plus Solanum nigrum-stimulated cells was almost completely inhibited by pretreatment with 100 micromol/l of pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappaB (NF-kappaB). Furthermore, Solanum nigrum increased activation of NF-kappaB. These findings suggest that Solanum nigrum increases the production of NO by rIFN-gamma-primed macrophages and NF-kappaB plays a critical role in mediating these effects.

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells.

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.

Effect of an extract of the root of Scutellaria baicalensis and its flavonoids on aflatoxin B1 oxidizing cytochrome P450 enzymes.

The inhibition of aflatoxin B1 (AFB1) metabolism by a water extract of the root of Scutellaria baicalensis and its flavonoids was examined in liver microsomes. AFB1 is known to be metabolized to aflatoxin M1 (AFM1), aflatoxin Q1 (AFQ1), and AFB1-8,9-epoxide (AFBO). The water extract potently inhibited the production of AFM1 by cytochrome P450 (CYP)1A1/2 and slightly reduced AFBO formation by CYP1A1/2, CYP2B1, CYP2C11 and CYP3A1/2 in TCDD-treated rat liver microsomes. IC50 values for AFM1 and AFBO formation were 6.8 and 122.4 microg/ml, respectively. Wogonin showed the highest inhibitory activity towards AFM1 formation among the flavonoids isolated from the extract. On the other hand, the extract had no effects on the formation of AFBO and AFQ1 in human liver microsomes, and on the activities of CYP2B1, CYP2C11 and CYP3A1/2 which were detected by hydroxylation patterns of testosterone. These results demonstrated that the extract of the root of Scutellaria baicalensis has a specific inhibitory effect on CYP1A1/2 among CYP enzymes involved in AFB1 metabolism by rat and human microsomes.

Activation of caspase cascades in Korean mistletoe (Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells.

Mistletoe lectins are of high biological activity and exert cytotoxic effects. We have previously shown that Korean mistletoe, Viscum album var. coloratum, lectin-II specifically induces apoptotic cell death in cancer cells, not normal lymphocytes. The destructive mechanism by mistletoe lectins on tumor cells was mediated by activation of c-JUN N-terminal kinase (JNK)/stress-activated protein kinase. Herein, we investigated the involvement of caspase cascade and its proteolytic cleavage effects on biosubstrates of human myeloleukemic U937 cells by D-galactoside and N-acetyl-galactosamine-specific Korean mistletoe lectin-II. Mistletoe lectin-II induced ladder pattern DNA fragmentation and activation of caspase-3, -8, and -9 of U937 cells, but not caspase-1 protease, in a time- and dose-dependent manner. Consistent with catalytic activation of protease, both poly(ADP-ribose) polymerase (PARP) and protein kinase C-delta (PKC-delta) are also cleaved in mistletoe lectin-II-treated U937 cells. An inhibitor of caspase-3-like protease, DEVD-CHO peptide, significantly inhibited mistletoe lectin-II-induced apoptosis, PARP cleavage, and fragmentation of DNA. These results provide the evidence that Korean mistletoe lectin-II induces apoptotic death of U937 cells via activation of caspase cascades.

Association of Shc, Cbl, Grb2, and Sos following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary rat hepatocytes.

c-Src kinases and p21 Ras are known to be implicated in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated signal transduction. However, the effects of TCDD on the molecular interaction of adaptor complex in the protein tyrosine kinase signaling cascade have not been reported. The present study is designed to clarify whether TCDD modulates the molecular interactions of Shc, Cbl, Grb2, and Sos in primary rat hepatocytes. TCDD causes an electrophoretic mobility shift of Sos and increases Sos/Grb2 association. Tyrosine phosphorylated Shc, mainly p52, unloads to the Grb2/Sos complex upon TCDD stimulation. Interestingly, TCDD decreases the tyrosine phosphorylation level of Cbl, although Cbl recruits more Grb2 and Shc proteins by TCDD. These results indicate that TCDD modulates the molecular interaction of adaptor complex proteins including Shc, Grb2, Sos, and Cbl. Furthermore, tyrosine phosphorylation of Cbl may not be critical for interaction of the protein with Grb2 and Shc in the TCDD signaling pathway in primary rat hepatocytes.