Dysregulation of PARP1 is involved in development of Barrett's esophagus

AIM To investigate the potential role of poly(ADP-ribose) polymerase 1(PARP1) in the development of Barrett's esophagus(BE).METHODS A BE mouse model was established to examine the esophageal morphological changes and molecular changes. Microarray analysis was performed to compare the gene expression profiles between BE patients and healthy controls. q PCR was used to examine the PARP1 expression in cell lines after treatment with H_2O_2 and bile acids(p H 4). Immunofluorescence staining, comet assay, and annexin V staining were used to evaluate the impact of PARP1 activity on cell survival and DNA damage response after oxidative stress.RESULTS The gene expression profile in normal and BE esophageal epithelial cells showed that PARP1, the major poly(ADP-ribose) polymerase, was overexpressed in BE. In the mouse model of BE, positive staining for NF-κB, γH2 AX, and poly(ADP-ribose)(PAR) was observed. H_2O_2 and bile acids(p H 4) increased the PARP1 m RNA expression level in normal esophageal epithelial cells. Using sh RNA-PARP1 to suppress PARP1 activity decreased the cell viability after treatment with H_2O_2 and bile acids(p H 4), and increased the oxidative damage as demonstrated by an increase in the levels of H_2O_2 , intracellular reactive oxygen species(ROS), oxidative DNA damage, double-strand breaks, and apoptosis(P 0.01).CONCLUSION The dysfunction of PARP1 in esophageal epithelial cells increases the levels of ROS and oxidative DNA damage, which could be common risk factors for BE and esophageal adenocarcinoma.     

Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bd-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells. METHODS: Gastric cancer cells of SGC-7901, MKN28, MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis indudng ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot. RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SC-C-7901cells respectively. Western blot revealed that the expressions of NF-EB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells. CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

High glucose augments stress-induced apoptosis in endothelial cells

Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. EAhy 926 endothelial ceils were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide （HzO2） or tumour necrosis factor- α （TNF- α ）, following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of EAhy 926 cells to apoptosis in the presence of 500 gmol/L H2O2 , above that induced in normal glucose （P〈0.02）. A reduction of H2O2- and TNF- a -induced apoptosis occurred in both high and low glucose after treatment with dexamethasone （P〈0.05）. Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.     

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway

AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance(TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respecti-vely used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. western blotting was also used to evaluate the cellular expression of myosin light chain(MLC), phosphorylated MLC(pM LC), MLC kinase(MLCK), and nuclear factor(NF)-κB p65. RESULTS TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cyto-kines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor(TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.     

Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene

Objective: To discuss the effect and molecular mechanism of mi R-146 a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor(MIF) gene. Methods: RT-PCR was employed to detect expression of mi R-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of mi R-146 a and the liposome LipofectamineTM2000 was employed to transfer the modeled mi R-146 a mimics, and mi R-146 a negative control(NC) in NSCLC cells to detect the expression of MIF m RNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, Annexin V-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3(Caspase 3), and western blot to detect expression of nuclear factor-κB(NF-κB) in cells. Results: The expression of mi R-146 a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of mi R-146 a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of mi R-146 a. The mi R-146 a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and m RNA was significantly decreased(P0.01), with the decrease in the cell viability(P0.01), the decrease in the number of clones(P0.01), cell apoptosis(P0.01), the increase in the activity of Caspase 3(P0.01), and decrease in the phosphorylation of NF-κB p65(P0.01). Conclusions: mi R-146 a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of mi R-146 a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.     

Rosiglitazone uppresses lipopolysaccharide-induced matrix metalloproteinase-2 activity in rat aortic endothelial cells via rasMEK1/2 signaling

ix metalloproteinase(MMPs) plays a key role in the pathogenesis of chronic inflammatory disease,such as atherosclerosis.Among MMPs,MMP-2 is regarded as a major proteinase in atherosclerotic plaque lesions.Peroxisome proliferator activated receptor-gamma(PPARg) ameliorates oxidative stress and the inflammatory response.The aim of the present study was to evaluate the effect of Rosiglitazone on Lipopolysaccharide(LPS)-induced MMP-2 activation as well as its possible mechanism.LPS-induced MMP-2 activity was inhibited by Rosiglitazone(PPARg agonist) in the rat aortic endothelial cells(RAEC).LPS-induced MMP-2 activation was diminished no matter exposure to NF-kB Activation Inhibitor II(JSH-23)or Ras inhibitor,farnesylthiosalicylic acid(FTS). Further study shows that LPS-induced activation of Phospho-Rho A and Phospho-MEKl/2 were significantly inhibited by Rosiglitazone.The activation of NF-kB p65 in the nuclear extract of cells was also significantly suppressed by Rosiglitazone, moreover,the expression of NF-κB p65 was partly activated by GW9662(PPARg antagonist).NF-kB DNA binding activity was also demolished by Rosiglitazone.In summary,our data showed that PPARg agonist,Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways and NF-kB activation.PPARg agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation.     

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase(ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480. METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA(si RNA) was used to knockdown expression of nuclear factor(NF)-κB/p65. Methylthiazole tetrazolium(MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition(EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B(IκB)a, inhibitor of gamma B(IγB)a, and nuclear factor kappa B(NF-κB) expressions and toexplore the ILK signaling pathway. RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells(P 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group(P 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed(P 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells(P 0.05). In order to determine the role of the NF-κB signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-κB/p65 and cytoplasmic phosphorylated-IκBa were increased and that cytoplasmic IкBa levels were decreased compared to the control group(P 0.05). Furthermore, NF-κB/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group. CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-κB signaling pathway.     

Both Atorvastatin and Probucol Can Down-regulate BMP-2 xpression Induced by Oxidized Low Density Lipoprotein in Human Umbilical Vein Cells

Bone morphogenetic protein-2 (BMP-2) plays an key role both in vascular development and pathophysiological processes. However, the mechanisms of oxidized low density lipoprotein (ox-LDL) and combinated with atorvastatin or probucol on BMP-2 expression are entirely unknown in human umbilical vein cells. Methods The HUVECs were treated by ox-LDL and combinated with atorvastatin, probucol. The expression of BMP-2, NF-κB65,PPARγ mRNA was examined by RT-PCR analysis and ELISA method. The MDA and SOD were detected by routine methods. Results Ox-LDL can induced BMP-2 mNRA expression, associated with NF-κB65 mNRA expression activation. Both atorvastatin and probucol can suppress BMP-2 and NF-κB65 expression induced by oxLDL and upregulate the expression of PPARγ. Furthermore, the increase of supernatant MDA levels and decrease of supernatant SOD levels resulted from oxLDL treatment can be reversed by probucol or atorvastatin. Conclusions OxLDL-induced BMP-2mNRA expression can be suppressed by atorvastatin and probucol, which may be accomplished by activating NF-κB65 expression and upregulating the expression of PPARγ. Our findings also indicate that that BMP-2 mNRA expression includes the activation of reactive oxygen species.     

Both Atorvastatin and Probucol Can Down-regulate BMP-2 Expression Induced by Oxidized Low Density Lipoprotein in Human Umbilical Vein Cells

Objectives Bone morphogenetic protein-2 (BMP-2) plays an key role both in vascular development and pathophysiological processes. However, the mechanisms of oxidized low density lipoprotein (ox-LDL) and combinated with atorvastatin or probucol on BMP-2 expression are entirely unknown in human umbilical vein cells. Methods The HUVECs were treated by ox-LDL and combinated with atorvastatin, probucol. The expression of BMP-2, NF-κB65, PPARγ mRNA was examined by RT-PCR analysis and ELISA method. The MDA and SOD were detected by routine methods. Results Ox-LDL can induced BMP-2 mNRA expression, associated with NF-κB65 mNRA expression activation. Both atorvastatin and probucol can suppress BMP-2 and NF-κB65 expression induced by oxLDL and upregulate the expression of PPARγ. Furthermore, the increase of supernatant MDA levels and decrease of supernatant SOD levels resulted from oxLDL treatment can be reversed by probucol or atorvastatin. Conclusions OxLDL-induced BMP-2 mNRA expression can be suppressed by atorvastatin and probucol, which may be accomplished by activating NF-κB65 expression and upregulating the expression of PPARγ. Our findings also indicate that that BMP-2 mNRA expression includes the activation of reactive oxygen species.