Blocking Protein Kinase Research Articles

Dexmedetomidine makes the anti-inflammation effect by blocking p38 mitogen-activated protein kinase in the lipopolysaccharides-induced acute lung injury in rats

Objective The aim of this study was to determine whether dexmedetomidine (Dex) could diminish lipopolysaccharide(LPS)-induced pulmonary injury by blocking lung p38 mitogen-activated protein kinase(p38MAPK) activation and inflammation in rats.Methods A total of 50 adult male SD rats were assigned to one of five groups:0.9％ sodium chloride group (C group),LPS group (LPS group) and LPS plus Dex groups (0.2,1,5 μg/kg per hour) (Dex0.2 group,Dex1 group and Dex5 group).The rats in the LPS plus Dex groups were injected a loading dose of Dex (1 μg/kg per hour,intravenous infusion over 10 minutes) followed by LPS administration and Dex infusion at different doses (0.2,1,5 μg/kg per hour,respectively) until the end of experiment.Simultaneously,the LPS and control groups received an equal volume of 0.9％ sodium chloride(1 ml/kg per hour) till the end of experiment.Western blot was used to detect the expression of phosphorylation of p38MAPK in lung tissues.Additionally,we examined the concentration of tumour necrosis factor-α(TNF-α) and interleukin(IL)-10 in bronchoalveolar lavage fluid(BALF),the histopathologic changes of lung,arterial blood gases and lung water content.Results With the administration of LPS,arterial oxygen partial pressure was significantly decreased (61.3±1.2) vs (131.7±4.7) (P＜0.05),Pathological examination showed that the normal structure of lung was destroyed badly after LPS injection,including intra-alveolar hemorrhage,interstitial edema,alveolar collapse and massive inflammatory cells infiltration,The lung water content was significantly increased (5.4±0.3) vs (3.6±0.2)(P＜ 0.05).Compared with the LPS group,lung water content of Dex5 group was significantly decreased(4.4±0.4) vs (5.4±0.3)(P＜0.05).With the administration of LPS,the phospho-p38 MAPK substantially increased immediately,and the concentrations of cytokines were also increased.However,Dex at the dose of 5.0 μg/kg per hour,but not at 0.2 or 1.0 μg/kg per hour,significantly attenuated these effects of LPS (P＜0.05).Conclusions Dex may attenuate LPS-induced acute lung injury via inhibiting p38MAPK activation and lung inflammation. Key words: Dexmedetomidine; Anti-inflammatory effect; p38 mitogen-activated protein kinase; Lipopolysaccharides; Acute lung injury

Participation of PI3K, MAPK ERK1/2, and p38 in the realization of growth potential of mesenchymal precursor cells under in vitro conditions.

We studied the role of intracellular signal molecules PI3K, MAPK ERK1/2, and p38 in the realization of the growth potential of mesenchymal progenitor elements. Under in vitro conditions, PI3K и ERK1/2 specifi c inhibitors reduced fi broblastic colony- and cluster-formation and considerably suppressed proliferative activity of mesenchymal precursors. Blocker of p38 and protein kinase B had no effect on the function of fi broblast CFU.

Increased Th17 Cells in the Tumor Microenvironment Is Mediated by IL-23 via Tumor-Secreted Prostaglandin E2

Tumor cell-derived molecules such as cytokines and lipid mediators play a critical role in inducing chronic inflammation in the tumor microenvironment. We found that Th17 cells were increased in the peripheral blood, spleen, and tumor tissues of mammary gland tumor-bearing mice. The Th17 cell survival factor, IL-23, was also overexpressed in tumor tissues isolated from mice and human breast cancer patients. Soluble molecules secreted from breast tumor cells, but not normal breast epithelial cells, induced IL-23 protein secretion in dendritic cells via induction of p19 mRNA expression. Our data further indicate that tumor-secreted PGE2 through EP2 and EP4 receptors enhanced IL-23 p19 gene transcription through binding to the cAMP-response element in the p19 promoter. Blocking PGE2 synthesis by NS398, a COX2 inhibitor, abrogated the enhancement of p19 expression both in vitro and in vivo. Furthermore, blocking protein kinase A (PKA) by H89 completely abrogated the inductive effects of tumor-conditioned medium and PGE2 on p19 transcription, whereas the cAMP active analog, Forskolin, mimics the PGE2 effect. Taken together, our results indicate that tumor-secreted PGE2 induces IL-23, but not IL-12, production in the tumor microenvironment, leading to Th17 cell expansion. This inductive effect of PGE2 on IL-23 p19 transcription is mediated through cAMP/PKA signaling transduction pathway.

Erratum: Corrigendum: ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system

Corrigendum: ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system

Regulation of Prostaglandin E2 signaling by cysteinyl leukotrienes in human mast cells

Cysteinyl leukotrienes (cys‐LTs), LTC4, LTD4 and LTE4, are critical mediators of airway hyper‐responsiveness and asthma. We have previously shown that LTD4 regulates mast cell (MCs) functions through the activation of G‐protein‐coupled receptors, CysLT1R and CysLT2R. The role of Prostaglandin E2 (PGE2), the other arachidonic acid metabolite in asthma is controversial as it act as both pro‐ and anti‐inflammatory, depending on the receptor through which it exerts its effect. Although cysLTs were shown to induce PGD2 generation in mast cells, it is not known whether there is cross‐talk between cysLTs and PGE2. Here, we found that PGE2 by itself does not activate calcium and MIP‐1β in mast cells, but cys‐LT priming induced robust PGE2‐dependent calcium flux and MIP‐1β generation. Further, using immunoprecipitation assays, we found that PGE2 receptor EP1R interacts with CysLT1R and this interaction was enhanced upon LTD4 stimulation. Importantly, we found that LTD4 and PGE2 synergistically increased the production of pro‐inflammatory cytokine, MIP1β in mast cells which was substantially inhibited by blocking protein kinase C (PKC)s. Taken together, our results suggest that cysLTs augment inflammation by activating PGE2 signaling through the priming of PGE2 receptors. This unravels a novel phenomenon by which inflammatory mediators generated from the same precursor can cooperate to enhance inflammation.

ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system

Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation.

Interaction of the Sympathetic Nerve with Pancreatic Cancer Cells Promotes Perineural Invasion through the Activation of STAT3 Signaling

Perineural invasion (PNI) is one of the most important causes of local recurrence and poor survival in pancreatic cancer. However, the exact mechanism of PNI is still not clear. In this study, we sought to identify the reciprocal signaling interactions between sympathetic nerves and pancreatic cancer cells and the underlying mechanisms. We used mouse dorsal root ganglia and pancreatic cancer cells cocultured in vitro, cellular and molecular biology, and animal models to evaluate the function of the sympathetic neurotransmitter norepinephrine (NE) in PNI progression and pathogenesis. NE promoted PNI of pancreatic cancer cells and increased levels of phosphorylated STAT3 in a concentration-dependent manner. NE-mediated activation of STAT3 was inhibited by blocking β-adrenergic receptors (AR) and by blocking protein kinase A, but not through blocking α-AR. Blocking STAT3 could inhibit NE-induced NGF, MMP2, and MMP9 expression and attenuate the migratory, invasive ability and PNI of pancreatic cancer cells. Furthermore, PNI of pancreatic cancer cells was blocked by treatment with a STAT3 phosphorylation inhibitor in vivo. These studies show that NE plays a critical role in pancreatic cancer PNI development and progression through the β-AR/PKA/STAT3 signaling pathway. Reciprocal signaling interactions between the sympathetic nerves and pancreatic cancer cells critically contribute to pancreatic cancer PNI pathogenesis. Inhibition of the activity of sympathetic nerves or STAT3 may be potential strategies for pancreatic cancer PNI therapy.

Blocking Protein kinase C signaling pathway: mechanistic insights into the anti-leishmanial activity of prospective herbal drugs from Withania somnifera

BackgroundLeishmaniasis is caused by several species of leishmania protozoan and is one of the major vector-born diseases after malaria and sleeping sickness. Toxicity of available drugs and drug resistance development by protozoa in recent years has made Leishmaniasis cure difficult and challenging. This urges the need to discover new antileishmanial-drug targets and antileishmanial-drug development.ResultsTertiary structure of leishmanial protein kinase C was predicted and found stable with a RMSD of 5.8Å during MD simulations. Natural compound withaferin A inhibited the predicted protein at its active site with -28.47 kcal/mol binding free energy. Withanone was also found to inhibit LPKC with good binding affinity of -22.57 kcal/mol. Both withaferin A and withanone were found stable within the binding pocket of predicted protein when MD simulations of ligand-bound protein complexes were carried out to examine the consistency of interactions between the two.ConclusionsLeishmanial protein kinase C (LPKC) has been identified as a potential target to develop drugs against Leishmaniasis. We modelled and refined the tertiary structure of LPKC using computational methods such as homology modelling and molecular dynamics simulations. This structure of LPKC was used to reveal mode of inhibition of two previous experimentally reported natural compounds from Withania somnifera - withaferin A and withanone.

An ECM-based culture system for the generation and maintenance of xeno-free human iPS cells

Pluripotent stem cells (PSCs) including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have emerged as a promising source for treating incurable diseases. Problems that urgently need to be resolved before the clinical application include avoiding potential xenopathogenic transmission and immune rejection that may be caused by the exposure of PSCs to animal-derived products. In addition, an efficient feeder cell-free culture condition would be required for reducing batch-to-batch variation and facilitating scale-up. Therefore, establishing an efficient xeno-free and extracelluar matrix-based culture system is a prerequisite for the clinical application of PSCs. In this study, by blocking protein kinase C and histone deacetylase activities, we formulated a medium that, in combination with vitronectin as an extracellular matrix, not only allows the long-term culture of hESCs and iPSCs but also efficiently generates xeno-free iPSCs. This xeno-free and feeder cell-free culture system would facilitate the clinical applications of both iPSC- and ESC-based cell therapies in the future.

Involvement of Basal and Calcium-Activated Protein Kinase C in Neurotransmitter Secretion in Mouse Motor Synapses

Blocker of presynaptic protein kinase C isoforms, GF109203X, reduced quantal content of single and rhythmic evoked end-plate potentials. The increase in quantal content of single potentials under the effect of 4- aminopyridine was neutralized by 75% under the effect of L-type Ca(2+)-channel blocker nitrendipine and completely returned to the control level after protein kinase C inhibition with chelerythrine. Neither nitrendipine, nor GF109203X affected the potentiating effect of tetraethylammonium on quantal content of end-plate potentials. Thus, we discovered basal activity of presynaptic protein kinase C under normal conditions that is aimed at the maintenance of quantal content of evoked release. It has been concluded that 4-aminipyridine, but not tetraethylammonium, triggers Ca(2+) entry into the terminal, which activates protein kinase C and enhanced the evoked acetylcholine release.

Rapid dopaminergic and GABAergic modulation of calcium and voltage transients in dendrites of prefrontal cortex pyramidal neurons

The physiological responses of dendrites to dopaminergic inputs are poorly understood and controversial. We applied dopamine on one dendritic branch while simultaneously monitoring action potentials (APs) from multiple dendrites using either calcium-sensitive dye, voltage-sensitive dye or both. Dopaminergic suppression of dendritic calcium transients was rapid (<0.5 s) and restricted to the site of dopamine application. Voltage waveforms of backpropagating APs were minimally altered in the same dendrites where dopamine was confirmed to cause large suppression of calcium signals, as determined by dual voltage and calcium imaging. The dopamine effects on dendritic calcium transients were fully mimicked by D1 agonists, partially reduced by D1 antagonist and completely insensitive to protein kinase blockade; consistent with a membrane delimited mechanism. This dopamine effect was unaltered in the presence of L-, R- and T-type calcium channel blockers. The somatic excitability (i.e. AP firing) was not affected by strong dopaminergic stimulation of dendrites. Dopamine and GABA were then sequentially applied on the same dendrite. In contrast to dopamine, the pulses of GABA prohibited AP backpropagation distally from the application site, even in neurons with natural Cl− concentration (patch pipette removed). Thus, the neocortex employs at least two distinct mechanisms (dopamine and GABA) for rapid modulation of dendritic calcium influx. The spatio-temporal pattern of dendritic calcium suppression described in this paper is expected to occur during phasic dopaminergic signalling, when midbrain dopaminergic neurons generate a transient (0.5 s) burst of APs in response to a salient event.

Histone deacetylase inhibitors enhance the apoptotic activity of insulin‐like growth factor binding protein‐3 by blocking PKC‐induced IGFBP‐3 degradation

Overexpression of insulin-like growth factor binding protein (IGFBP)-3 induces apoptosis of cancer cells. However, preexisting resistance to IGFBP-3 could limit its antitumor activities. This study characterizes the efficacy and mechanism of the combination of recombinant IGFBP-3 (rIGFBP-3) and HDAC inhibitors to overcome IGFBP-3 resistance in a subset of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells. The effects of the combination of rIGFBP-3 and a number of HDAC inhibitors on cell proliferation and apoptosis were assessed in vitro and in vivo by using the MTT assay, a flow cytometry-based TUNEL assay, Western blot analyses and the NSCLC xenograft tumor model. Combined treatment with HDAC inhibitors and rIGFBP-3 had synergistic antiproliferative effects accompanied by increased apoptosis rates in a subset of NSCLC and HNSCC cell lines in vitro. Moreover, combined treatment with depsipeptide and rIGFBP-3 completely suppressed tumor growth and increased the apoptosis rate in vivo in H1299 NSCLC xenografts. Evidence suggests that HDAC inhibitors increased the half-life of rIGFBP-3 protein by blocking protein kinase C (PKC)-mediated phosphorylation and degradation of rIGFBP-3. In addition, combined treatment of IGFBP-3 with an HDAC inhibitor facilitates apoptosis through upregulation of rIGFBP-3 stability and Akt signaling inhibition. The ability of HDAC inhibitors to decrease PKC activation may enhance apoptotic activities of rIGFBP-3 in NSCLC cells in vitro and in vivo. These results indicated that combined treatment with HDAC inhibitor and rIGFBP-3 could be an effective treatment strategy for NSCLC and HNSCC with highly activated PKC.

Sphingosine-1-phosphate induces contraction of valvular interstitial cells from porcine aortic valves

Sphingosine-1-phosphate (S1P) has emerged as a potent bioactive lipid with multiple functions in cardiovascular pathophysiology. Potential roles of S1P in heart valve diseases and expression of relevant receptors (S1P1, S1P2, or S1P3) in valve tissue and in valvular interstitial cells (VICs), the major cell population with essential functions in maintenance of valvular structure, are currently unknown. Exposure to S1P (62-2000 nM) of cultured VICs from porcine aortic valves on cell culture polystyrene resulted in contraction and nodule formation. The S1P-dependent contraction was completely inhibited by blockers of S1P2, RhoA, and RhoA-associated protein kinase (ROCK). Activated RhoA was clearly increased after S1P treatment, whereas activated Rac1 was only slightly reduced. In addition, exposure to S1P induced a transient increase in cytosolic Ca(2+). Application of channel blockers and other effectors of Ca(2+) homeostasis showed that the S1P effect is largely caused by Ca(2+) release from internal stores. However, resistance to blocking S1P2, different kinetics, as well as concentration dependence exclude a major role of Ca(2+) influx in S1P-induced nodule formation. In order to verify the effects in situ, contractions of valve tissue slices were measured. The S1P-induced isometric contraction of valve leaflets was of similar force amplitude as observed with adrenaline. The effect was fully reversed by blocking S1P2. The results suggest that S1P induces contraction of VICs from porcine aortic valves by signalling via S1P2, RhoA, and ROCK. In this way, S1P may contribute to regulation of tissue tension in aortic valves.

The afterhyperpolarizing potential following a train of action potentials is suppressed in an acute epilepsy model in the rat Cornu Ammonis 1 area

In hippocampal Cornu Ammonis 1 (CA1) neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP), which critically dampens neuronal excitability. Because it is not known whether epileptiform activity alters the AHP and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABA A receptor blocker gabazine (5 μM) in the rat hippocampal slice preparation and studied its impact on the AHP using intracellular recordings. Following 10 min of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 min of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 μM) and d-(−)-2-amino-5-phosphonopentanoic acid ( d-AP5, 50 μM) to block α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N-methyl- d-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABA A receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 μM) and H-89 (1 μM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process—presumably involving protein kinase A.

Stimulation of Ca2 +-sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney

We used the patch-clamp technique to study the effect of changing the external Ca2+ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2+ concentration from 1mM to 2 or 3mM inhibited the basolateral 50-pS K channels while decreasing external Ca2+ to 10μM increased the 50-pS K channel activity. The effect of the external Ca2+ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2+ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2+-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2+ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analog increased the 50-pS K channel activity but did not block the effect of raising the external Ca2+ on the K channels. Neither inhibition of phospholipase A2 (PLA2) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2+ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2+. We conclude that the external Ca2+ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2+ is mediated by the CaSR which stimulates PLC–PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2+ level modulates the reabsorption of divalent cations.

New drugs beyond biologics in rheumatoid arthritis: the kinase inhibitors

Orally available small molecule compounds have recently been developed for the treatment of RA, and inhibitors of signalling cascades, specifically inhibitors of kinases, have reached advanced stages of clinical development. The p38 mitogen-activated protein kinase blockers have shown poor clinical response despite encouraging preclinical data. In contrast, inhibitors of the non-receptor tyrosine kinases, spleen tyrosine kinase and janus kinase 3, have demonstrated a significant clinical efficacy together with an acceptable safety profile. We herein present a review on published preclinical and clinical data on these new drugs.

Nitric Oxide-mediated Relaxation by High K+in Human Gastric Longitudinal Smooth Muscle

This study was designed to elucidate high-K+induced response of circular and longitudinal smooth muscle from human gastric corpus using isometric contraction. Contraction from circular and longitudinal muscle stripes of gastric corpus greater curvature and lesser curvature were compared. Circular smooth muscle from corpus greater curvature showed high K+ (50 mM)-induced tonic contraction. On the contrary, however, longitudinal smooth muscle strips showed high K+ (50 mM)-induced sustained relaxation. To find out the reason for the discrepancy we tested several relaxation mechanisms. Protein kinase blockers like KT5720, PKA inhibitor, and KT5823, PKG inhibitor, did not affect high K+-induced relaxation. K+ channel blockers like tetraethylammonium (TEA), apamin (APA), glibenclamide (Glib) and barium (Ba2+) also had no effect. However, N(G)-nitro-L-arginine (L-NNA) and 1H-(1,2,4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC) and 4-AP (4-aminopyridine), voltage-dependent K+ channel (KV) blocker, inhibited high K+-induced relaxation, hence reversing to tonic contraction. High K+-induced relaxation was observed in gastric corpus of human stomach, but only in the longitudinal muscles from greater curvature not lesser curvature. L-NNA, ODQ and KV channel blocker sensitive high K+-induced relaxation in longitudinal muscle of higher portion of corpus was also observed. These results suggest that longitudinal smooth muscle from greater curvature of gastric corpus produced high K+-induced relaxation which was activated by NO/sGC pathway and by KV channel dependent mechanism.

Targeted therapies in diabetic nephropathy: an update

Diabetic nephropathy is the most common cause of end-stage renal disease worldwide. Various pathways in addition to the renin-angiotensinogen system have been implicated in the pathogenesis of diabetic nephropathy. Strategies to interrupt these pathophysiological pathways are a key to the development of new targeted therapies to prevent progression of diabetic nephropathy and are on the horizon. The various pharmacological drugs tried include aliskiren, a direct renin inhibitor blocking the first step in the renin pathway, and pentoxifylline and mammalian target of rapamycin inhibitors, which have antiinflammatory properties and have shown some promising results in management of diabetic nephropathy. Others include endothelin antagonists and vitamin D analogs which have been shown to decrease urinary albumin excretion. Inhibitors of advanced glycation and oxidative stress and plasminogen activator inhibitor-1 have proved useful in animal models of diabetic nephropathy. Others include ruboxistaurin, which blocks protein kinase C overexpression. Such targeted therapies would halt and might even reverse progression of diabetic nephropathy.

Progesterone Rapidly Attenuates ATP-Evoked Transient Currents in Cultured Rat Dorsal Root Ganglion Neurons

Background: Progesterone has been shown to play a role in pain perception. However, the effects of progesterone on P2X3 receptors, the nociception-related receptors in primary sensory neurons, remain unclear. Methods: We investigated the effects of progesterone on P2X3-receptor-mediated responses of rat dorsal root ganglion neurons by using whole-cell patch clamp techniques. Results: Progesterone (10 pmol/l to 1 µmol/l) inhibited ATP (100 µmol/l)-induced transient currents and the transient component of biphasic currents in a dose-dependent manner, but had no effect on the sustained currents. The effect of progesterone could be blocked by its receptor inhibitor, RU38486. Actidione, an inhibitor of protein synthesis, did not change the rapid effect. Investigations of the signaling pathways of progesterone showed that a nonselective antagonist of protein kinase, H-9, totally blocked the depressive effect of progesterone on ATP currents in a dose-dependent manner. Blocking protein kinase A with H-89 or KT5720 also affected progesterone-induced depression. The protein kinase A activator, forskolin, mimicked the effect of progesterone on ATP currents. Conclusion: These results suggest that progesterone may modulate pain signal transmission on dorsal root ganglia via regulating P2X3 receptor function. The cAMP-PKA signaling pathway is involved in the downregulating effect of progesterone on P2X3 receptors.

Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules.

Background:Enzastaurin (Enz) is a serine/threonine kinase inhibitor blocking protein kinase C (PKC)β/AKT pathway. However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated.Methods:The ability of Enz to inhibit invasion and metastasis, and to target molecules was investigated in non-small cell lung cancer (NSCLC) by RT–PCR validated microarray, Matrigel, and in vivo chorionallantoic membrane (CAM) assays.Results:Enzastaurin significantly reduced migration, invasion, and in vivo metastasis to lungs and liver (CAM assay) of diverse NSCLC cell lines. Genes promoting cancer progression (u-PAR, VEGFC, and HIF1α) and tumour suppression (VHL, RASSF1, and FHIT) of NSCLC were significantly (P<0.05) down- or upregulated after Enz treatment in H460, A549, and H1299 cells, respectively. Luciferase/chromatin immunoprecipitation analysis showed that Enz transcriptionally controls urokinase-type plasminogen activator receptor (u-PAR) expression by promoter inhibition through Sp1, Sp3, and c-Jun(AP-1). Moreover, siRNA knockdown of u-PAR re-sensitised Enz-resistant cells and induced apoptosis, suggesting u-PAR as a marker of Enz resistance.Conclusion:This study shows that Enz inhibits migration, invasion, and in vivo metastasis by targeting u-PAR, besides further targeting progression-related and tumour-suppressor genes in NSCLC. Together with u-PAR being a novel putative marker of Enz response, these data encourage molecularly tailored clinical studies on Enz in NSCLC therapy.