Antidepressant mechanism and active compounds of saffron from network pharmacology study.

Saffron has been applied in depression treatment, but its antidepressant compounds and mechanisms are unclear. In this research, a network pharmacology-based method was proposed to screen the active compounds and the potential mechanisms of saffron for depression treatment. Firstly, the chemical compounds of saffron were collected from literature and filtered by drug-like prediction. Secondly, common targets, by comparing the targets of saffron predicted by Pharm Mapper server with targets associated with depression collected from Genecards, were regarded as the antidepressant targets of saffron. Thirdly, common targets were mapped to KEGG pathways, considered as the pathways related with the antidepressant effects of saffron. Finally, the network of compounds-targets-pathways was constructed and analyzed by cytoscape 3.4.0. Ten compounds including crocetin, picrocrocin, (1R, 5S, 6R)-5-(hydroxymethyl)- 4, 4, 6-trimethyl-7-Oxabicyclo[4.1.0]heptan-2-one and its glycoside were screened as the main antidepressant compounds, some of which were reported for the first time. They might have effective treatment for depression by acting on targets, such as MAP2K1, MAPK1, HRAS, PIK3R1, ALB and AKT1 and pathways related with immune system, signal transduction and so on. This study provided a new insight into the antidepressant mechanism and active compounds of saffron, which also had a guiding effect on later experiments.

Factor Xa induces tissue factor expression in endothelial cells by P44/42 MAPK and NF-κB-dependent pathways.

BACKGROUND: Tissue factor (TF) is an initiator of coagulation. The serine protease factor Xa (FXa) is the convergence point of the extrinsic and intrinsic components of the coagulation cascade. In addition to its hemostatic function, FXa elicits inflammatory responses in endothelial cells that may be important in surgical procedures in which inflammation is triggered. This study tested the hypothesis that FXa can up-regulate TF on vascular endothelial cells by a mitogen-activated protein kinase (MAPK)- and NF-κB-dependent pathway. METHODS AND RESULTS: Incubation of cultured human umbilical vein endothelial cells (HUVECs) with FXa increased TF protein expression and activity in a dose-dependent manner. Pre-incubation of HUVECs with the serine protease inhibitor antithrombin, which targets not only thrombin but also FXa and FIXa, inhibited FXa-induced TF expression, but the selective thrombin inhibitor hirudin did not inhibit FXa-induced TF expression, ruling out a thrombin-mediated pathway. After 10 min incubation with HUVECs, FXa rapidly induced P44/42 MAPK activation (immunoblotting of phosphorylated P44/42 MAPK) with a peak at 30 min. The MEK 1/2 inhibitor PD98059 partially reduced FXa-induced TF expression and activity (3.82 ± 0.11 vs 6.54 ± 0.08 fmol/min/cm(2), P < 0.05). NF-κB was activated by FXa, confirmed by cytoplasmic IkBα degradation and increased NF-κB P65 nuclear translocation. Interruption of the NF-κB pathway by the IkBα phosphorylation inhibitor Bay 11-7802 abrogated FXa-induced TF protein expression and activity (1.93 ± 0.02 versus 6.54 ± 0.08 fmol/min/cm(2), P < 0.05). However, inhibition of PI3 kinase by LY 294002 did not attenuate FXa-induced TF protein expression and activity. CONCLUSIONS: (1) FXa up-regulates TF protein expression and activity in HUVECs, (2) FXa-induced up-regulation of TF is independent of the thrombin-PAR1 pathway, and (3) the MAPK and NF-κB pathways, but not PI3 kinase pathway, are involved in FXa-induced TF expression on human umbilical endothelial cells. FXa may be a feed-forward alternative mechanism of activating TF expression and activity, thereby increasing a procoagulant state or inflammation. This mechanism may be important in the pro-inflammatory state initiated by cardiac surgical procedures.

Response to BRAF and MEK1/2 inhibition in a young adult with BRAF V600E mutant epithelioid glioblastoma multiforme: A Case Report and Literature Review.

Epithelioid glioblastoma multiforme (eGBM) is a rare and aggressive variant of glioblastoma multiforme (GBM) that predominantly affects younger patients and can be difficult to distinguish from other gliomas. Data on how patients with eGBM might be best treated are limited, although genomic analyses have shown that almost half of tumours harbour activating BRAF gene mutations. Here we present the case of a young female with BRAF V600E-mutant eGBM who had a prolonged response to targeted therapy with the BRAF and MEK1/2 inhibitors dabrafenib and trametinib. We review current knowledge about eGBM, including the emerging role for BRAF- ± MEK1/2- targeted therapy.

Role of MEK-ERK pathway in morphine-induced conditioned place preference in ventral tegmental area of rats.

A major goal of research on drug addiction is to develop the effective treatments to deal with the long-term behavioral disorders especially reinstatement induced by the addictive drugs such as opiates, cocaine, and cannabinoid. The molecular mechanisms underlying these substance-related disorders remain unclear so far. Here we used the model of morphine-induced conditioned place preference (CPP) in rats to mimic the progress of drug-taking, withdrawal and relapse in human. The tissue of ventral tegmental area (VTA), one of the most important brain structures associated with abused drug-related disorders, was taken and two-dimensional electrophoresis (2-DE) was performed to analyze and compare the changes of protein expression patterns during the different stages of morphine-induced CPP. First, we found that there were 80 proteins identified to be changed in the process of morphine-induced CPP. Furthermore, as the mitogen-activated protein kinase kinase 1 (MAPKK1) was increased significantly in the stages of establishment and reinstatement, we confirmed the change of activated extracellular signal-regulated kinase (ERK) by Western blotting in VTA tissue and cultured cell. The results demonstrated that the activated MEK-ERK pathway by chronic morphine treatment in VTA was involved in morphine-induced reinstatement. Moreover, inhibition of MEK-ERK pathway by infusion the MEK inhibitor U0126 in VTA blocked the establishment of morphine-induced CPP. The present study found significant changes in a group of protein expressions in VTA during morphine-induced CPP and further confirmed the role of MEK-ERK cell signaling pathway of VTA in morphine addiction.

Activity regulates positive and negative neurotrophin-derived signals to determine axon competition.

Developmental axon competition plays a key role in sculpting neural circuitry. Here, we have asked how activity and neurotrophins could interact to select one axon over another. Using compartmented cultures of sympathetic neurons, we show that, in the presence of NGF, local depolarization confers a competitive growth advantage on the depolarized axon collaterals and at the same time disadvantages the growth of unstimulated axons from the same and competing neurons. Depolarization mediates the competitive advantage by activating a CaMKII-MEK pathway, which converges to enhance local NGF-mediated downstream growth signals. Patterned electrical stimulation also acts via this pathway to enhance NGF-promoted axonal growth. In contrast, the competitive disadvantage is due to BDNF secreted from and acting on the unstimulated, competing axons through p75NTR. Thus, activity regulates both positive and negative neurotrophin-derived signaling cascades to confer a competitive growth advantage on one axon versus another, thereby providing a cellular mechanism for developmental axon selection.

Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities.

AIMS: Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. METHODS AND RESULTS: Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). CONCLUSION: Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

A chemical biology approach identifies a beta-2 adrenergic receptor agonist that causes human tumor regression by blocking the Raf-1/Mek-1/Erk1/2 pathway.

A chemical biology approach identifies a beta 2 adrenergic receptor (beta2AR) agonist ARA-211 (Pirbuterol), which causes apoptosis and human tumor regression in animal models. beta2AR stimulation of cAMP formation and protein kinase A (PKA) activation leads to Raf-1 (but not B-Raf) kinase inactivation, inhibition of Mek-1 kinase and decreased phospho-extracellular signal-regulated kinase (Erk)1/2 levels. ARA-211 inhibition of the Raf/Mek/Erk1/2 pathway is mediated by PKA and not exchange protein activated by cAMP (EPAC). ARA-211 is selective and suppresses P-Erk1/2 but not P-JNK, P-p38, P-Akt or P-STAT3 levels. beta2AR stimulation results in inhibition of anchorage-dependent and -independent growth, induction of apoptosis in vitro and tumor regression in vivo. beta2AR antagonists and constitutively active Mek-1 rescue from the effects of ARA-211, demonstrating that beta2AR stimulation and Mek kinase inhibition are required for ARA-211 antitumor activity. Furthermore, suppression of growth occurs only in human tumors where ARA-211 induces cAMP formation and decreases P-Erk1/2 levels. Thus, beta2AR stimulation results in significant suppression of malignant transformation in cancers where it blocks the Raf-1/Mek-1/Erk1/2 pathway by a cAMP-dependent activation of PKA but not EPAC.

Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell.

In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.

MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100 particle assembly.

OBJECTIVE: Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway. METHODS AND RESULTS: HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins. CONCLUSIONS: Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor.

PURPOSE: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. EXPERIMENTAL DESIGN: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. RESULTS: The IC(50) of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor-induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate-induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. CONCLUSIONS: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.

Mu opioid receptor mutant, T394A, abolishes opioid-mediated adenylyl cyclase superactivation.

This study was to characterize the effects of a point-mutant at C-terminal of mu opioid receptor (MOR), namely MOR T394A, in chronic opioid-induced cellular responses. After 18 h of exposure to [D-Ala, N-Me-Phe, Gly-ol] enkephalin (DAMGO), adenylyl cyclase (AC) superactivation, a hallmark for the cellular adaptive response after chronic opioid stimulation, was observed in the cells expressing wild-type receptor, but was totally abolished in the cells expressing MOR T394A. Receptor phosphorylation was also attenuated in cells with MOR T394A after prolonged preexposure to agonist. Furthermore, MAP kinase kinase-1 (MKK1) overexpression was able to rescue AC superactivation in cells with MOR T394A, but showed no effect in the wild-type MOR-expressing cells. These results indicated that the amino acid T394 at C-terminus of MOR played a critical role in chronic agonist-induced AC superactivation and receptor phosphorylation.

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.

Sulindac independently modulates extracellular signal-regulated kinase 1/2 and cyclic GMP-dependent protein kinase signaling pathways.

Colorectal cancer is the second leading cause of cancer mortality in the United States. Substantial human and animal data support the ability of nonsteroidal anti-inflammatory drugs to cause regression of existing colon tumors and prevent new tumor formation. The mechanism by which the nonsteroidal anti-inflammatory drug sulindac prevents tumor growth is poorly understood and seems complex as sulindac can modulate several growth-related signaling pathways. Sulindac metabolites simultaneously (a) increase cellular cyclic GMP and subsequently activate cyclic GMP-dependent protein kinase (PKG); (b) activate c-jun NH2-terminal kinase (JNK); (c) inhibit extracellular signal-regulated kinase 1/2 (ERK1/2); and (d) decrease beta-catenin protein expression at times and doses consistent with apoptosis. The purpose of this study was to determine if PKG, ERK1/2, JNK, and beta-catenin are independent targets for sulindac in vitro. Pharmacologic activation of PKG with YC-1 increases JNK phosphorylation and induces apoptosis in colon cancer cells without modulating ERK1/2 phosphorylation or beta-catenin protein expression. Inhibition of ERK1/2 with U0126 induces apoptosis but fails to activate JNK phosphorylation or down-regulate beta-catenin protein expression. Cotreatment with U0126 and YC-1 synergistically increases apoptosis in colorectal cancer cells and recapitulates the effects of sulindac treatment on ERK1/2, JNK, and beta-catenin. These results indicate that sulindac metabolites modulate ERK1/2 and PKG pathways independently in colon cancer cells and suggest that the full apoptotic effect of sulindac is mediated by more than one pathway. Using similar combinatorial approaches in vivo may provide more effective, less toxic chemopreventive and chemotherapeutic strategies. Such therapies could dramatically reduce the incidence and death rate from colorectal cancer.

p90RSK Blockade Inhibits Dual BRAF and MEK Inhibitor-Resistant Melanoma by Targeting Protein Synthesis.

Despite improvements in survival in metastatic melanoma with combined BRAF and mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor treatment, the overwhelming majority of patients eventually acquire resistance to both agents. Consequently, new targets for therapy in resistant tumors are currently being evaluated. Previous studies have identified p90 subfamily of ribosomal S6 kinase (p90RSK) family kinases as key factors for growth and proliferation, as well as protein synthesis via assembly of the 7-methyl-guanosine triphosphate cap-dependent translation complex. We sought to evaluate inhibitors of p90RSK family members: BI-D1870 and BRD7389, for their ability to inhibit both proliferation and protein synthesis in patient-derived melanoma cell lines with acquired resistance to combined treatment with the BRAF inhibitor vemurafenib and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor selumetinib. We found that the RSK inhibitors blocked cell proliferation and protein synthesis in multiple dual-resistant melanoma lines. In addition, single agent RSK inhibitor treatment was effective in drug-naïve lines, two of which are innately vemurafenib resistant. We also used Reverse Phase Protein Array screening to identify differential protein expression that correlates with BI-D1870 sensitivity, and identified prognostic biomarkers for survival in human melanoma patients. These findings establish p90RSK inhibition as a therapeutic strategy in treatment-resistant melanoma and provide insight into the mechanism of action.

Inhibitors of the mitogen-activated protein kinase kinase 1/2 signaling pathway clear prion-infected cells from PrPSc.

Prions represent a unique class of infectious agents in which the normal cellular prion protein (PrPC) is converted to an abnormal isoform (PrPSc), which accumulates in the brain and constitutes the major, if not the only, component of the infectious particle. Factors that still remain to be identified may facilitate the conversion of PrPC to PrPSc. In the present study, we first demonstrated that a growth factor of the neurotrophin family, brain-derived neurotrophic factor (BDNF), stimulates the formation of PrPSc in a gonadotropin-releasing hormone-secreting neuronal cell line (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie as determined by Western blot analysis. We then observed that the prion-infected cells can be cleared from PrPSc by treatment with three inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene and 2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one, as well as alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile, which passes the blood-brain barrier], a component of one of the intracellular signaling pathways activated by BDNF. The MEK1/2 inhibitors were also efficient in clearing PrPSc from prion-infected GT1-1 cells stimulated to accumulate high levels of PrPSc by enhanced serum concentrations in the medium or by the use of a serum-free neuron-specific neurobasal medium. PrPSc did not reappear in the cultures within 5 weeks after completion of treatment. We conclude that inhibitors of the MEK1/2 pathway can efficiently and probably irreversibly clear PrP(Sc) from prion-infected cells. The MEK pathway may therefore be a suitable target for therapeutic intervention in prion diseases.

Methamphetamine causes alterations in the MAP kinase-related pathways in the brains of mice that display increased aggressiveness.

Aggressive behaviors have been reported in patients who suffer from some psychiatric disorders, and are common in methamphetamine (METH) abusers. Herein, we report that multiple (but not single) injections of METH significantly increased aggressiveness in male CD-1 mice. This increase in aggressiveness was not secondary to METH-induced hyperactivity. Analysis of protein expression using antibody microarrays and Western blotting revealed differential changes in MAP kinase-related pathways after multiple and single METH injections. There were statistically significant (p<0.05) decreases in MEK1, Erk2p, GSK3alpha, 14-3-3e, and MEK7 in the striata of mice after multiple injections of METH. MEK1 was significantly decreased also after a single injection of METH, but to a much lesser degree than after multiple injections of METH. In the frontal cortex, there was a statistically significant decrease in GSK3alpha after multiple (but not single) injections of METH. These findings suggest that alterations in MAP kinase-related pathways in the prefronto-striatal circuitries might be involved in the manifestation of aggressive behaviors in mice.

COX-2 inhibitors suppress lung cancer cell growth by inducing p21 via COX-2 independent signals.

COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. The mechanisms by which COX-2 exerts its mitogenic effects have not been entirely elucidated, but stimulation of prostaglandin E2 production and alterations in the expression of the cyclin-dependent kinase inhibitor p21(WAF-1/CIP1/MDA-6)(p2i) have been suggested. Here, we demonstrate that two COX-2 inhibitors (NS398 and Nimesulide) inhibit proliferation and induce apoptosis in NSCLC cells, and these effects were associated with induction of p21 mRNA and protein expression. However, the anti-growth effect of the COX-2 inhibitors and their ability to induce p21 were not affected by COX-2 siRNA suggesting that their actions were COX-2 independent. Instead, activation of the MEK-1/Erk pathway was necessary since COX-2 inhibitors stimulated the phosphorylation of ERKs, and their effects were blocked by PD98095, an inhibitor of this pathway. Furthermore, we show that both NS398 and Nimesulide induced p21 gene promoter activity and this was prevented by PD98095. COX-2 inhibitors increased nuclear protein binding to the Spl site in the promoter region of the p21 gene. Consistent with a role for p21, we found that p21 antisense oligonucleotides prevented the effects of COX-2 inhibitors on cell growth. In summary, our results suggest that COX-2 inhibitors suppress NSCLC cell growth by inducing the expression of the p21 gene through MEK-1/ERK signaling and DNA-protein interactions involving Spl. These observations unveil a mechanism for p21 gene regulation by COX-2 inhibitors in lung carcinoma cell growth and this pathway represents a potential target for therapy.

Inhibition of vascular smooth muscle cell proliferation by Gentiana lutea root extracts.

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.