Chi Li Chung

Nuclear-targeted inhibition of NF-κB on MMP-9 production by N-2-(4-bromophenyl) ethyl caffeamide in human monocytic cells

Aberrant remodeling of the extracellular matrix occurs in many pathological processes, and its breakdown is mainly accomplished by matrix metalloproteinases (MMPs), which participate in the course of inflammation and tumor invasion. Nuclear factor-kappaB (NF-kappaB), a key transcription factor for the production of MMP-9, can be activated by various proinflammatory cytokines and promotes inflammation. In the present study, we investigated the intracellular mechanism for the inhibitory effects of an analogue of N-hydroxycinnamoylphenalkylamides, N-2-(4-bromophenyl) ethyl caffeamide (EK5), on tumor necrosis factor (TNF)-alpha stimulated expression of MMP-9 in a human monocytic cell line, THP-1. Our results show that TNF-alpha-induced expression of MMP-9 at both mRNA and protein levels was completely blocked by EK5 in a concentration-dependent (1-20microM) manner. We also found that EK5 markedly suppressed NF-kappaB signaling as detected by the NF-kappaB reporter gene assay but had no effects on the degradation of IkappaBalpha or translocation of NF-kappaB. Interestingly, chromatin immunoprecipitation results revealed that the association between p65 and MMP-9 promoter gene was completely abrogated by EK5, but the p65 phosphorylation was not affected. Overall, our findings suggest that EK5 inhibits MMP-9 production through the nuclear-targeted down-regulation of NF-kappaB signaling in human monocytic cells and this may provide a novel molecular basis of EK5 activity. Further studies are needed to verify its anti-inflammatory effects.

Dynasore, a dynamin inhibitor, induces PAI-1 expression in MeT-5A human pleural mesothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is a primary regulator of plasminogen activation that plays an essential role in regulating the physiological thrombotic/fibrinogenic balance. The elevation of PAI-1 expression by human pleural mesothelial cells has been reported to contribute to pleural fibrosis and pleurodesis. In this study, we examined the effects on PAI-1 expression of dynasore, a cell-permeable inhibitor of dynamin, and its mechanisms in a human pleural mesothelial cell line (MeT-5A). The results indicated that dynasore enhanced transforming growth factor (TGF)-beta(1)- and TNF-alpha-induced PAI-1 protein expression in a concentration-dependent manner. Furthermore, dynasore significantly up-regulated PAI-1 protein and its messenger RNA expressions. Interestingly, Smad2/3 activation was induced by TGF-beta(1) but not by dynasore. Among signaling inhibitors, a c-Jun NH(2)-terminal kinase (JNK) inhibitor (SP600125) markedly attenuated dynasore-stimulated PAI-1 protein production. Consistently, dynasore strongly increased JNK phosphorylation. On the other hand, there was no enhancement effect by dynasore on TGF-beta(1)-induced matrix metalloproteinase-2 activation. These findings suggest that dynasore may stimulate PAI-1 protein expression and enhance TGF-beta(1) activity through activation of JNK-mediated signaling in human pleural mesothelial cells. Given the profibrotic effect of dynasore, further in vivo studies may be conducted to evaluate its potential as a pleurodesing agent.

Suppression of matrix metalloproteinase-9 expression by andrographolide in human monocytic THP-1 cells via inhibition of NF-κB activation.

There is much evidence indicating that human leukemic cells and monocytes/macrophages synthesize, and secrete, several matrix metalloproteinases (MMPs), and participate in the degradation of extracellular matrix components in tissue lesions. In this study, we investigated the effects and mechanisms of andrographolide, extracted from the herb Andrographis paniculata, on human monocytic MMPs expression and activation. Andrographolide (1-50 μM) exhibited concentration-dependent inhibition of MMP-9 activation, induced by either tumor necrosis factor-α (TNF-α), or lipopolysaccharide (LPS), in THP-1cells. In addition, andrographolide did not present an inhibitory effect on MMP-9 enzymatic activity at a concentration of 50 μM. By contrast, enzyme-linked immunosorbent assay (ELISA) showed that andrographolide partially affect TIMP-1 levels. Western blot analysis showed that both TNF-α, and LPS stimulators attenuated MMP-9 protein expression in a concentration-dependent manner. Using reverse transcription polymerase chain reaction (RT-PCR), we found that andrographolide suppressed expression of MMP-9 messenger RNA. Furthermore, we also found that andrographolide could significantly inhibit the degradation of inhibitor-κB-α (IκB-α) induced by TNF-α. We used electrophoretic mobility shift assay and reporter gene detection to show that andrographolide also markedly inhibited NF-κB signaling, anti-translocation and anti-activation. In conclusion, we demonstrate that andrographolide attenuates MMP-9 expression, and its main mechanism might involve the NF-κB signal pathway. These results provide new opportunities for the development of new anti-inflammatory and leukemic therapies.

Impact of epidermal growth factor receptor mutations on intracranial treatment response and survival after brain metastases in lung adenocarcinoma patients

INTRODUCTION Brain metastases (BM) commonly occur in patients with lung adenocarcinoma and usually lead to a poor prognosis and quality of life despite of radiotherapy. Epidermal growth factor receptor (EGFR) mutations have been widely demonstrated to be a predictive and prognostic factor for lung adenocarcinoma, however, its impact on BM from lung adenocarcinoma remains inconclusive. The present study aimed to elucidate the predictive role of EGFR mutations in BM treatment response and survival after BM in patients with lung adenocarcinoma. MATERIAL AND METHODS From January 2006 through February 2012, 180 of 505 lung adenocarcinoma patients developed BM during their disease course were reviewed for eligibility, and 139 patients, including 89 EGFR-mutant and 50 EGFR wild-type patients, were identified for analysis. RESULTS Of the patients eligible for evaluation of treatment response, up to 85% received radiotherapy and the remaining took EGFR tyrosine kinase inhibitors (TKIs) as the front modality for BM. EGFR-mutant patients, compared with EGFR wild-type patients, had significantly greater intracranial treatment response of BM (84% vs. 48%, P = 0.002), experienced higher therapeutic efficacy to radiotherapy (86% vs. 52%, P = 0.005), and had longer median survival after BM diagnosis (13.2 vs. 6.8 months, P < 0.001). Furthermore, EGFR mutation (P = 0.002) and performance status (P = 0.009) were independently associated with BM treatment response. Additionally, EGFR mutation (P = 0.005), good performance status (P < 0.001) and absence of extracranial metastases (P=0.033) correlated with better survival. CONCLUSION EGFR mutation is an independent predictive factor for both BM treatment response and survival after BM in patients with lung adenocarcinoma. Further prospective studies on incorporation of EGFR mutation status into therapeutic strategy and survival prediction system for lung adenocarcinoma with BM are warranted.

Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo

The multidrug resistance protein 4 (MRP4) is a member of the ABCC subfamily of the adenosine triphosphate-binding cassette transporters that remove cyclic nucleotides from platelets and uptake ADP into dense granule in platelets. However, whether MRP4 directly involves platelet activation remains unclear. Thus, the aim of our study was to determine the detailed mechanisms underlying the regulation of MRP4 in platelet activation. Our results revealed that the MRP4 inhibitor MK571 inhibited collagen-induced platelet aggregation which was partially reversed by the PKA inhibitor H89, but not by the adenylyl cyclase (AC) inhibitor SQ22536 and the guanylyl cyclase (GC) inhibitor ODQ, suggesting that MK571 can prevent collagen-induced aggregation via a route independent of cyclic nucleotide production. In the present study, we found that MK571 inhibited collagen-induced ATP release and calcium mobilization. The phosphorylation of protein kinase C, JNK, and Akt was also inhibited by MK571, and electron spin resonance experiment showed that MK571 significantly reduced hydroxyl radical formation. Moreover, MK571 delayed platelet plug formation in vitro by a PFA-100 device, and delayed thrombus formation in mesenteric venules of mice irradiated by fluorescein sodium. However, previous studies have reported that MK571 also blocks MRP1 and leukotriene D4 (LTD4) receptor. Therefore, whether MK571 inhibits platelet activation through MRP1 or LTD4 receptor needs to be considered and further defined. In conclusion, in addition to blocking the transport of cyclic nucleotides, MRP4 inhibition may prevent thrombus formation in vitro and in vivo. Our findings also support the idea that MRP4 may represent a potential target for the development of novel therapeutic interventions for the treatment of thromboembolic disorders.

Suitability of computed tomography-guided biopsy specimens for subtyping and genotyping of non-small-cell lung cancer

INTRODUCTION Recent advances in the treatment of NSCLC highlight the importance of distinguishing NSCLC subtypes and genotypes. We aimed to determine whether histological specimens obtained from computed tomography (CT)-guided biopsy are suitable for specific subtyping and epidermal growth factor receptor (EGFR) analyses of NSCLC. PATIENTS AND METHODS The clinicohistological data of 332 consecutive patients undergoing 352 CT-guided biopsies for lung lesions between January 2007 and December 2011 were retrospectively analyzed. Additionally, NSCLC specimens were examined for the suitability of EGFR mutational testing. RESULTS Of 209 specimens diagnosed as NSCLC, 197 (94.3%) were specifically subtyped into adenocarcinoma (n = 164; 78.5%), squamous cell carcinoma (n = 27; 12.9%) and other subtypes (n = 6; 2.9%). The rate of NSCLC not otherwise specified (NOS) was 5.7%, and the diagnosis of NSCLC-NOS was significantly associated with the poor differentiation of cancer (adjusted odds ratio, 6.17; 95% confidence interval, 1.62-23.55; P = .008). Of 134 histological tumor specimens submitted for EGFR molecular testing, 132 (98.5%) were suitable for analyses, and 130 of them (98.5%) showed conclusive results, revealing 59.8% (n = 79) with EGFR exon mutation(s). The sensitivity, specificity, and positive and negative predictive values of CT-guided biopsy in patients with malignancy were 92.2%, 100%, 100%, and 74.1%, respectively. Six percent (n = 21) of total lung biopsies led to pneumothorax requiring chest drainage, and no procedure-related fatality was observed. CONCLUSION Small tumor specimens obtained with CT-guided needle lung biopsy are suitable for specific subtyping and EGFR analyses of NSCLC, thus providing critical information for personalized therapy.

Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

BackgroundBrazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.MethodsIn this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.ResultsWe demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin αIIbβ3 in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α2β1, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.ConclusionTo the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.

Pleural space elastance and changes in oxygenation after therapeutic thoracentesis in ventilated patients with heart failure and transudative pleural effusions

Background and objective:  Therapeutic thoracentesis (TT) is required in patients with refractory pleural effusions and impaired oxygenation. In this study, the relationship between pleural space elastance (PE) and changes in oxygenation after TT was investigated in ventilated patients with heart failure and transudative pleural effusions.

Repeated Thoracenteses Affect Proinflammatory Cytokines, Vascular Endothelial Growth Factor, and Fibrinolytic Activity in Pleural Transudates

Background:Repeated thoracenteses is indicated in patients with refractory, symptomatic transudative effusions. However, their effect on cytokines and fibrinolytic activity in pleural transudates remains unclear. Methods:Twenty-one patients with symptomatic, large amount of free-flowing transudative effusions caused by heart failure were studied. Thoracentesis with drainage of 500 mL of pleural fluid per day was done for 3 consecutive days (days 1 to 3). Pleural fluid characteristics, tumor necrosis factor (TNF)-&agr;, interleukin (IL)-1&bgr;, IL-8, vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured during each tap. Chest ultrasonography was done on day 6 to detect the fibrin strands in pleural effusion and the outcome of effusion was evaluated within 7 days after repeated thoracenteses. Results:Effusion levels of lactate dehydrogenase, neutrophils, TNF-&agr;, IL-1&bgr;, IL-8, VEGF, and PAI-1 increased significantly during repeated thoracenteses. Furthermore, the values of PAI-1 and PAI-1/tPA obtained on days 2 and 3 were highly correlated with those of TNF-&agr;, IL-1&bgr;, IL-8, and VEGF. On day 6, pleural fibrins were observed on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of TNF-&agr;, IL-1&bgr;, VEGF, and PAI-1 on day 2 and day 3, and recurrence of symptomatic effusion after repeated thoracenteses were significantly higher in fibrinous group. Conclusions:Repeated thoracenteses may induce local release of proinflammatory cytokines, VEGF and PAI-1, which may result in fibrin deposition and impair resolution of pleural transudates.

Mycobacterium tuberculosis Upregulates TNF-α Expression via TLR2/ERK Signaling and Induces MMP-1 and MMP-9 Production in Human Pleural Mesothelial Cells

Background Tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) are elevated in pleural fluids of tuberculous pleuritis (TBP) where pleural mesothelial cells (PMCs) conduct the first-line defense against Mycobacterium tuberculosis (MTB). However, the clinical implication of TNF-α and MMPs in TBP and the response of PMCs to MTB infection remain unclear. Methods We measured pleural fluid levels of TNF-α and MMPs in patients with TBP (n = 18) or heart failure (n = 18) as controls. Radiological scores for initial effusion amount and residual pleural fibrosis at 6-month follow-up were assessed. In vitro human PMC experiments were performed to assess the effect of heat-killed M. tuberculosis H37Ra (MTBRa) on the expression of TNF-α and MMPs. Results As compared with controls, the effusion levels of TNF-α, MMP-1 and MMP-9 were significantly higher and correlated positively with initial effusion amount in patients with TBP, while TNF-α and MMP-1, but not MMP-9, were positively associated with residual pleural fibrosis of TBP. Moreover, effusion levels of TNF-α had positive correlation with those of MMP-1 and MMP-9 in TBP. In cultured PMCs, MTBRa enhanced TLR2 and TLR4 expression, activated ERK signaling, and upregulated TNF-α mRNA and protein expression. Furthermore, knockdown of TLR2, but not TLR4, significantly inhibited ERK phosphorylation and TNF-α expression. Additionally, both MTBRa and TNF-α markedly induced MMP-1 and MMP-9 synthesis in human PMCs, and TNF-α neutralization substantially reduced the production of MMP-1, but not MMP-9, in response to MTBRa stimulation. Conclusion MTBRa activates TLR2/ERK signalings to induce TNF-α and elicit MMP-1 and MMP-9 in human PMCs, which are associated with effusion volume and pleural fibrosis and may contribute to pathogenesis of TBP. Further investigation of manipulation of TNF-α and MMP expression in pleural mesothelium may provide new insights into the mechanisms and rational treatment strategies for TBP.