Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.     

Lps induces IL-6 in the brain and in serum largely through TNF production

We investigated the relative contribution of IL-6 and PGE2 directly induced by LPS and indirectly induced via TNF, using in vivo and in vitro models in the mouse. In these models we have used as tools an anti-TNF antibody and a cyclooxygenase inhibitor, the S enantiomer of ketoprofen (S-KPF). Anti-TNF antibodies inhibited LPS-induced IL-6 production in three different models: IL-6 production by mouse peritoneal macrophages in vitro; serum IL-6 levels induced by intraperitoneal LPS; and brain IL-6 levels induced by an intracerebroventricular injection of LPS. However, in vitro anti-TNF antibodies, did not inhibit LPS-induced PGE2, indicating that this effect is not mediated by TNF. Since PGE2 has an opposite effect on TNF and IL-6 production, inhibiting that of TNF but inducing that of IL-6, we investigated the effect of S-KPF on TNF and IL-6 production in vivo following LPS injection. Both TNF and IL-6 induction was augmented by S-KPF, but anti-TNF antibodies abolished the augmentation of IL-6 production. Thus, the effect of anti-inflammatory drugs on IL-6 production in some models can be secondary to their effect on TNF production.     

Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate

The development of LPS tolerance has been suggested to be mediated by an inhibition of cytokine synthesis. Here we have studied serum IL-6 and TNF levels in mice after LPS administration. Repeated administration of LPS (35 micrograms daily for 4 days) to mice induced a refractoriness (tolerance) to subsequent administrations of LPS in terms of induction of circulating IL-6 and TNF. To investigate the mechanism by which LPS down-regulates its own induction of cytokine synthesis and the relationship between IL-6 and TNF production, we attempted to revert the inhibition of IL-6 and TNF production using agents like PMA or IFN-gamma, previously reported to activate macrophage production of cytokines. Pretreatment with PMA (4 micrograms, 10 min before LPS) partially restored IL-6 production in LPS-tolerant mice given 2 micrograms LPS. On the other hand, PMA did not restore TNF induction in LPS-tolerant mice, even when administered with high doses of LPS (up to 200 micrograms). A similar reversal of LPS resistance to IL-6, but not TNF, induction by PMA was observed in genetically LPS-resistant C3H/HeJ mice. IFN-gamma also restored, although to a lesser extent than PMA, IL-6 production. However, unlike PMA, IFN-gamma could also partially restore TNF production in LPS-tolerant mice, although only when LPS was administered at high doses. By contrast with PMA, IFN-gamma was clearly more active in restoring TNF synthesis than that of IL-6. Similar results were obtained in genetically LPS-unresponsive C3H/HeJ mice. These data suggest that different mechanisms are implicated in the inhibition of IL-6 and TNF synthesis in LPS-tolerant mice and that part of this inhibition can be overcome by PMA or IFN-gamma.     

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200 mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.     

IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice

Incubation of the human U937 histiocytic lymphoma cell line with granulocyte-macrophage colony stimulating factor (GM-CSF) rendered the cells responsive to induction of TNF by LPS. Treatment with IL-6 reduced TNF production in GM-CSF-primed U937 cells. The inhibitory effect was most pronounced (approximately equal to 80%) when IL-6 was added either along with GM-CSF or within the first 3 h of GM-CSF treatment. Both GM-CSF or IL-6 inhibited [3H]TdR uptake in U937 cells, and simultaneous treatment with GM-CSF and IL-6 resulted in an additive inhibitory effect on cell proliferation. However, the inhibition of TNF production could not be explained by the inhibitory effect of IL-6 on cell growth, nor was it due to a reduction in cell viability. An inhibition of TNF production by IL-6 was also demonstrated in cultured human peripheral blood monocytes. Treatment with IL-6 also resulted in a dose-dependent reduction of the 17-kDa TNF band revealed by SDS-PAGE after labeling monocytes with [35S]cysteine and immunoprecipitation with anti-TNF mAb. In addition, treatment with IL-6 resulted in a reduction of monocyte in vitro cytotoxicity for tumor target cells. Finally, in mice sensitized by the administration of Bacillus Calmette-Guérin, the injection of IL-6 significantly reduced the levels of TNF found in the serum upon challenge with LPS. Inasmuch as TNF is known to be an inducer of IL-6, the inhibitory action of IL-6 on TNF production may represent the negative arm of a regulatory circuit. The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role of IL-6 in the intact organism.     

R-isomers of Arg-Gly-Asp (RGD) mimics as potent alphavbeta3 inhibitors

The integrin alpha(v)beta(3), vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. We recently disclosed the discovery of a tripeptide Arg-Gly-Asp (RGD) mimic, which has been shown to be a potent inhibitor of the integrin alpha(v)beta(3) and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In other investigations involving RGD mimics, only compounds containing the S-isomers of the beta-amino acids have been shown to be potent. We were surprised to find the potencies of analogs containing enantiomerically pure S-isomers of beta-amino acids which were only marginally better than the corresponding racemic mixtures. We therefore synthesized RGD mimics containing R-isomers of beta-amino acids and found them to be relatively potent inhibitors of alpha(v)beta(3). One of the compounds was examined in tumor models in mice and has been shown to significantly reduce the rate of growth and the size of tumors.     

CDX2 can be regulated through the signalling pathways activated by IL-6 in gastric cells

The inflammatory infiltrate of the gastric mucosa associated with Helicobacter pylori infection increases the presence of the pro-inflammatory cytokine IL-6 that activates both the SHP-2/ERK/MAPK and the JAK/STAT signalling pathways. Furthermore, the ectopic expression of CDX2 is detected in pre-neoplasic lesions associated with decreased levels of SOX2, and we found that in gastric adenocarcinomas their expression is inversely correlated. To determine the role of IL-6 in the regulation of CDX2, MKN45 that constitutively expresses p-STAT3, and NUGC-4 gastric cancer cell lines were treated with IL-6, which induced the CDX2 up-regulation and SOX2 down-regulation. ChIP assays determined that in IL-6-treated cells, c-JUN and p-STAT3 bound to CDX2 promoter in MKN45 cells whereas in NUGC-4 cells, p-STAT3 binds to and c-JUN releases from the CDX2 promoter. Specific inhibition of STAT3 and ERK1/2 phosphorylation through AG490 and U0126, respectively, and STAT3 down-regulation using shRNA verified that the SHP-2/ERK/MAPK pathway regulates the expression of CDX2 in basal conditions, and the CDX2 up-regulation by IL-6 is through the JAK/STAT pathway in NUGC-4 cells whereas in MKN45 cells both pathways contribute to the CDX2 up-regulation. In conclusion, the signalling pathways activated by IL-6 have a crucial role in the regulation of CDX2 that is a key factor in the process of gastric carcinogenesis, suggesting that the inflammatory infiltrate in the gastric mucosa is relevant in this process and a potential target for new therapeutic approaches.     

Granulocyte colony-stimulating factor decreases tumor necrosis factor production in whole blood: role of interleukin-10 and prostaglandin E(2)

Previous reports have indicated that the administration of granulocyte colony-stimulating factor (G-CSF) decreases ex vivo tumor necrosis factor (TNF) production in humans. In this study, we report that daily pretreatment of mice with G-CSF for three days decreases ex vivo lipopolysaccharide (LPS)-induced TNF production in whole blood. Conversely, production of interleukin-10 (IL-10) and prostaglandin E(2) (PGE(2)) is increased. The inhibitory effect of G-CSF pretreatment on TNF production is partially reversed by addition of an anti-IL-10 antibody, and completely reversed by combined addition of anti-IL-10 antibody and the cyclooxygenase (COX) inhibitor, ketoprofen. These results suggest that G-CSF decreases TNF production in this experimental model by increasing production of IL-10 and PGE(2), which are both known inhibitors of TNF production.     

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

Selection of novel analogs of thalidomide with enhanced tumor necrosis factor alpha inhibitory activity.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNF alpha production such as tuberculous meningitis or toxic shock.     

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.     

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology.

Interleukin-10 (IL-10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL-10-deficient mice requires the function of TNF, indicating that the IL-10/TNF axis regulates mucosal immunity. We further show that IL-10 targets the 3' AU-rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL-10 does not alter TNF mRNA stability, and its action does not require the presence of the stability-regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL-10 is exerted mainly by inhibition of the activating p38/MAPK-activated protein kinase-2 pathway. These results demonstrate a physiologically significant cross-talk between the IL-10 receptor and the stress-activated protein kinase modules targeting TNF mRNA translation. This cross-talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut.     

Inhibition of TNF-induced IL-6 by the TWEAK-Fn14 interaction in rheumatoid arthritis fibroblast like synoviocytes

ObjectivesTNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.MethodsRAFLS were obtained from surgical synovial specimens and used at passage 5–10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.ResultsTWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.ConclusionsTWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.     

S-adenosylmethionine (SAMe) modulates interleukin-10 and interleukin-6, but not TNF, production via the adenosine (A2) receptor

S-adenosylmethionine (SAMe) is the first product in methionine metabolism and serves as a precursor for glutathione (GSH) as well as a methyl donor in most transmethylation reactions. The administration of exogenous SAMe has beneficial effects in many types of liver diseases. One mechanism for the hepatoprotective action is its ability to regulate the immune system by modulating cytokine production from LPS stimulated monocytes. In the present study, we investigated possible mechanism(s) by which exogenous SAMe supplementation modulated production of TNF, IL-10 and IL-6 in LPS stimulated RAW 264.7 cells, a murine monocyte cell line. Our results demonstrated that exogenous SAMe supplementation inhibited TNF production but enhanced both IL-10 and IL-6 production. SAMe increased intracellular GSH level, however, N-acetylcysteine (NAC), the GSH pro-drug, decreased the production of all three cytokines. Importantly, SAMe increased intracellular adenosine levels, and exogenous adenosine supplementation had effects similar to SAMe on TNF, IL-10 and IL-6 production. 3-Deaza-adenosine (DZA), a specific inhibitor of S-adenosylhomocysteine (SAH) hydrolase, blocked the elevation of IL-10 and IL-6 production induced by SAMe, which was rescued by the addition of exogenous adenosine. Furthermore, the enhancement of LPS-stimulated IL-10 and IL-6 production by both SAMe and adenosine was inhibited by ZM241385, a specific antagonist of the adenosine (A(2)) receptor. Our results suggest that increased adenosine levels with subsequent binding to the A(2) receptor account, at least in part, for SAMe modulation of IL-10 and IL-6, but not TNF production, from LPS stimulated monocytes.