Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀ = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5-5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and beta-adrenergic receptor signaling pathways

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE₂ and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE₂ induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

Activation of prostaglandin EP receptors by lubiprostone in rat and human stomach and colon

BACKGROUND AND PURPOSE: Lubiprostone (Amitiza), a possible ClC-2 channel opener derived from prostaglandin E(1) and indicated for the treatment of constipation, increases chloride ion transport and fluid secretion into the intestinal lumen. As lubiprostone may also directly modulate gastrointestinal motility, we investigated its actions and the possible involvement of prostaglandin EP receptor activation on rat and human isolated gastrointestinal preparations. EXPERIMENTAL APPROACH: Rat and human isolated preparations were mounted in tissue baths for isometric recording. The effects of lubiprostone on muscle tension and on electrically stimulated, neuronal contractions were investigated in the absence and presence of EP receptor antagonists. KEY RESULTS: In rat and human stomach longitudinal muscle, lubiprostone induced a contraction (pEC(50) of 7.0+/-0.0, n=4 and 6.4+/-0.2, n=3, respectively), which was inhibited by pretreatment with the EP(1) receptor antagonist, EP(1)A 300 nM (pEC(50) reduced to 6.2+/-0.2, n=6), but not by the EP(3) or EP(4) receptor antagonists (L-798106 and GW627368X, respectively, 1 microM, P>0.05). Lubiprostone also reduced electrically stimulated, neuronal contractions in rat and human colon circular muscle preparations (pIC(50) of 8.9+/-0.4, n=7 and 8.7+/-0.9, n=6, respectively), an effect mediated pre-junctionally. This effect was reduced by the EP(4) receptor antagonist (pIC(50) of 6.7+/-1.1, n=7 and 7.7+/-0.4, n=6, respectively) but not by EP(1) or EP(3) receptor antagonists. CONCLUSIONS AND IMPLICATIONS: In rats and humans, lubiprostone contracts stomach longitudinal muscle and inhibits neuronally mediated contractions of colon circular muscle. Experiments are now needed to determine if this additional activity of lubiprostone contributes to its clinical efficacy and/or side-effect profile.     

UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells

The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5–25 mJ/cm²). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.     

Role of Ca2+-independent phospholipase A2 in cell growth and signaling

Phospholipase A₂ (PLA₂) are esterases that cleave glycerophospholipids to release fatty acids and lysophospholipids. Several studies demonstrate that PLA₂ regulate growth and signaling in several cell types. However, few of these studies have focused on Ca²⁺-independent phospholipase A₂ (iPLA₂ or Group VI PLA₂). This class of PLA₂ was originally suggested to mediate phospholipid remodeling in several cell types including macrophages. As such, it was labeled as a housekeeping protein and thought not to play as significant of roles in cell growth as its older counterparts cytosolic PLA₂ (cPLA₂ or Group IV PLA₂) and secretory PLA₂ (sPLA₂ or Groups I-III, V and IX-XIV PLA₂). However, several recent studies demonstrate that iPLA₂ mediate cell growth, and do so by participating in signal transduction pathways that include epidermal growth factor receptors (EGFR), mitogen activated protein kinases (MAPK), mdm2, and even the tumor suppressor protein p53 and the cell cycle regulator p21. The exact mechanism by which iPLA₂ mediates these pathways are not known, but likely involve the generation of lipid signals such as arachidonic acid, lysophosphatidic acid (LPA) and lysophosphocholines (LPC). This review discusses the role of iPLA₂ in cell growth with special emphasis placed on their role in cell signaling. The putative lipid signals involved are also discussed.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells

Hypoxia appears to induce a program which shifts the cellular phenotype toward an increase in extracellular adenosine. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. Since in gliomas there is a strong correlation between HIF-1alpha expression, tumor grade and tumor vascularization, the aim of this study was to investigate whether adenosine may regulate HIF-1 in human glioblastoma cell lines. The results indicate that in the human hypoxic A172 and U87MG glioblastoma cell lines adenosine up-regulates HIF-1alpha protein expression via the A(3) receptor subtype. In particular, we investigated the effect of A(3) receptor antagonists on HIF-1 and vascular endothelial growth factor (VEGF) expression. We found that A(3) antagonists inhibit adenosine-induced HIF-1alpha and VEGF protein accumulation in the hypoxic cells. Investigations in the molecular mechanism showed that A(3) receptor stimulation activates p44/p42 and p38 MAPKs that are required for A(3)-induced increase of HIF-1alpha and VEGF. Further studies are required to demonstrate the in vivo relevance of these observations with regard to the proposed role for adenosine as a key element in hypoxia and in tumors.     

Stimulation of early gene induction and cell proliferation by lysophosphatidic acid in human amnion-derived WISH cells: role of phospholipase D-mediated pathway

Human-amniotic WISH cells express the lysophosphatidic acid (LPA) receptor, LPA(1), LPA(2) but not LPA(3). When WISH cells were stimulated with LPA, phospholipase D (PLD) activation was dramatically induced via a cytosolic calcium increase and protein kinase C activation. We also found that LPA stimulated two kinds of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38 kinase via PLD-dependent signaling pathways in WISH cells. In terms of the LPA-mediated functional modulation of WISH cells, we observed that LPA stimulates the induction of two early genes (c-Jun and c-Fos) and cellular proliferation in WISH cells. We examined the signaling pathways involved in LPA-mediated cellular responses. LPA-induced early gene induction was completely blocked by normal butanol (n-butanol) but not by t-butanol, suggesting that PLD activity is essentially required for the process. PD98059 (2'-amino-3'-methoxyflavone) but not SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) also significantly blocked LPA-induced early gene induction, suggesting a crucial role for ERK. Pertussis toxin (PTX) did not affect on the LPA-induced early gene induction and ERK activation, ruling out the role of Gi/o protein(s) in the process. The cellular proliferation of WISH cells was also dramatically inhibited by n-butanol or PD98059. This study demonstrates the physiological role of LPA on the modulation of early gene induction and on WISH cell proliferation, and the crucial role played by PLD in the process.     

Differential production of leukotriene B4 or prostaglandin E2 by WKYMVm or serum amyloid A via formyl peptide receptor-like 1

Serum amyloid A (SAA) and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) have been reported as formyl peptide receptor-like 1 (FPRL1) ligands. WKYMVm but not SAA stimulated superoxide generation by human neutrophils. In terms of the downstream signalings triggered by WKYMVm and SAA, both agonists stimulated cytosolic phospholipase A2-mediated arachidonic acid release, a precursor of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). WKYMVm also strongly stimulated LTB4 production in human neutrophils without affecting PGE2 production, whereas SAA strongly stimulates cyclooxygenase-2 (COX-2) expression and PGE2 production but not LTB4 production. In terms of the receptors responsible for the differential actions of these two agonists, we found that FPRL1 is involved in the production of LTB4 by WKYMVm and PGE2 production by SAA. This study demonstrates that the chemoattractant receptor, FPRL1, can be differentially regulated by distinct ligands to generate different lipid mediators, and thus, different immune responses.     

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.     

Inhibition of osteoclastogenesis by a phosphodiesterase 4 inhibitor XT-611 through synergistic action with endogenous prostaglandin E2

We examined the effect of a phosphodiesterase 4 (PDE4) inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611) on osteoclast formation in three different mouse bone-marrow cell (BMC) culture systems. We confirmed that selective inhibitors of PDE4, including XT-611, among several PDE inhibitors decreased osteoclast formation in the BMC culture system. XT-611 also inhibited osteoclast formation in co-culture of mouse bone-marrow stromal cell line ST2 and adherent cell-depleted (ACD)-BMCs. However, it did not inhibit osteoclastogenesis in culture of ACD-BMCs alone in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-kappaB ligand (sRANKL). XT-611 significantly increased prostaglandin E(2) (PGE(2)) production from ST2 cells and, in combination with PGE(2), synergistically increased cAMP concentration in osteoclast progenitors. In the ST2 co-culture system, XT-611 did not influence the expression of RANKL, osteoprotegerin and RANK mRNAs. By combined treatment with XT-611 and PGE(2) of ACD-BMCs, osteoclast multinucleation was clearly inhibited with decrease in the expression of calcitonin receptor mRNA, while the expression of RANK and c-fms (an M-CSF receptor) mRNAs was unchanged. These results indicate that the PDE4 inhibitor inhibits osteoclastogenesis by acting on osteoclast progenitors synergistically with PGE(2) secreted from stromal cells, but not by influencing the cell-to-cell interaction between stromal cells and osteoclast progenitors.     

Direct mitochondrial dysfunction precedes reactive oxygen species production in amiodarone-induced toxicity in human peripheral lung epithelial HPL1A cells

Amiodarone (AM), a drug used in the treatment of cardiac dysrrhythmias, can produce severe pulmonary adverse effects, including fibrosis. Although the pathogenesis of AM-induced pulmonary toxicity (AIPT) is not clearly understood, several hypotheses have been advanced, including increased inflammatory mediator release, mitochondrial dysfunction, and free-radical formation. The hypothesis that AM induces formation of reactive oxygen species (ROS) was tested in an in vitro model relevant for AIPT. Human peripheral lung epithelial HPL1A cells, as surrogates for target cells in AIPT, were susceptible to the toxicity of AM and N-desethylamiodarone (DEA), a major AM metabolite. Longer incubations (> or =6 h) of HPL1A cells with 100 microM AM significantly increased ROS formation. In contrast, shorter incubations (2 h) of HPL1A cells with AM resulted in mitochondrial dysfunction and cytoplasmic cytochrome c translocation. Preexposure of HPL1A cells to ubiquinone and alpha-tocopherol was more effective than that with Trolox C or 5,5-dimethylpyrolidine N-oxide (DMPO) at preventing AM cytotoxicity. These data suggest that mitochondrial dysfunction, rather than ROS overproduction, represents an early event in AM-induced toxicity in peripheral lung epithelial cells that may be relevant for triggering AIPT, and antioxidants that target mitochondria may potentially have beneficial effects in AIPT.     

Molecular mechanisms of ZD1839-induced G1-cell cycle arrest and apoptosis in human lung adenocarcinoma A549 cells

Epithelial growth factor receptor (EGFR) has been proposed as a target for anticancer therapy. ZD1839 (Iressa) is a quinazoline derivative that selectively inhibits the EGFR tyrosine kinase activity and is under clinical use in cancer patients. However, the molecular mechanisms involved in ZD1839-mediated anticancer effects remain largely uncharacterized. In this study, exposure of human lung adenocarcinoma A549 cells to ZD1839 caused G1 arrest, and subsequently induced apoptosis. Moreover, ZD1839 increased the protein levels of p27(KIP1) and retinoblastoma-related Rb2/p130 while decreased the expression of cyclin-dependent kinase-2 (CDK2), CDK4, CDK6 and cyclin-D1, cyclin-D3. In vitro kinase assay showed that ZD1839 decreased these CDKs expression in A549 cells, leading to significantly reduce their kinase activities. In addition, ZD1839-induced death of A549 cells with characteristics of apoptosis including apoptotic morphological changes, DNA fragmentation and enhancement of TUNEL-positive cell. These events were accompanied by a marked increase of Fas protein expression, and activation of caspase-2, -3, -8. Co-treatment of cells with Fas antagonist antibody significantly blocked ZD1839-induced apoptosis. Caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, were also capable of restoring cell viability. Our results indicate that downregulation of the expression and function of CDK2, CDK4, CDK6, cyclin-D1 and cyclin-D3, as well as upregulation of p27(KIP1) and pRb2/p130, are strong candidates for the cell cycle regulator that arrests ZD1839-treated A549 cells at G1 phase. Furthermore, upregulation of Fas appears to play a major role in the initiation of ZD1839-induced apoptosis, activation of caspase-8/caspase-3 cascade is involved in the execution phase of this death program.     

Novel amidine analogue of melphalan as a specific multifunctional inhibitor of growth and metabolism of human breast cancer cells

A novel amidine analogue of melphalan (AB4) was compared to its parent drug, melphalan in respect to cytotoxicity, DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. It was found that AB4 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than melphalan. The topoisomerase I/II inhibition assay indicated that AB4 is a potent catalytic inhibitor of topoisomerase II. Data from the ethidium displacement assay showed that AB4 intercalated into the minor-groove at AT sequences of DNA. The greater potency of AB4 to suppress collagen synthesis was found to be accompanied by a stronger inhibition of prolidase activity and expression compared to melphalan. The phenomenon was related to the inhibition of beta(1)-integrin and IGF-I receptor mediated signaling caused by AB4. The expression of beta(1)-integrin receptor, as well as Sos-1 and phosphorylated MAPK, ERK(1) and ERK(2) but not FAK, Shc, and Grb-2 was significantly decreased in cells incubated for 24h with 20 microM AB4 compared to the control, not treated cells, whereas in the same conditions melphalan did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the amidine analogue of melphalan, AB4 represent multifunctional inhibitor of breast cancer cells growth and metabolism.     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.