u-0126 and Esophageal-Neoplasms

Histone deacetylases (HDACs) have been demonstrated to be aberrantly activated in tumorigenesis and cancer development. Thus, HDAC inhibitors (HDACIs) are considered to be promising anti-cancer therapeutics. However, recent studies have shown that HDACIs promote the migration of many cancer cells. Therefore, there is a need to elucidate the underlying mechanisms of HDACIs on cancer cell migration to establish a combination therapy that overcomes HDACI-induced cell migration.. KYSE-150 and EC9706 cells were treated differently. Effects of drugs and siRNA treatment on tumor cell migration and cell signaling pathways were investigated by transwell migration assy. Gene expression for SNAI2 was tested by RT-qPCR. Western blot analysis was employed to detect the level of E-cadherin, β-catenin, vimentin,Slug，ERK1/2, H3, PAI-1 and BRD4. The effect of drugs on cell morphology was evaluated through phase-contrast microscopic images.. TSA promotes epithelial-mesenchymal transition (EMT) in ESCC cells by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers β-catenin, vimentin, Slug, and PAI-1. Knockdown of Slug by siRNA or inhibition of PAI-1 clearly suppressed TSA-induced ESCC cell migration and resulted in the reversal of TSA-triggered E-cadherin, β-catenin, and vimentin expression. However, no crosstalk between Slug and PAI-1 was observed in TSA-treated ESCC cells. Blocking ERK1/2 activation also inhibited TSA-induced ESCC cell migration, EMT, and upregulation of Slug and PAI-1 levels in ESCC cells. Interestingly, inhibition of BRD4 suppressed TSA-induced ESCC cell migration and attenuated TSA-induced ERK1/2 activation and upregulation of Slug and PAI-1 levels.. Our data indicate the existence of at least two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration: an ERK1/2-Slug branch and an ERK1/2-PAI-1 branch. Both branches of TSA-induced ESCC cell migration appear to favor the EMT process, while BRD4 is responsible for two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration.

Topics: beta Catenin; Butadienes; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Shape; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flavonoids; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Nitriles; Plasminogen Activator Inhibitor 1; Protein Kinase Inhibitors; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Vimentin

Esophageal carcinoma is a frequently occurring cancer at upper gastrointestinal tract. We aimed to evaluate the roles and possible mechanism of Runt Related Transcription Factor 2 (RUNX2) in the development of esophageal cancer.. The expression of RUNX2 in esophageal carcinoma tissues and cells was investigated by qRT-PCR. Effects of RUNX2 on cell viability, apoptosis, migration and invasion were assessed using MTT assay, flow cytometry assay/western blot analysis, and Transwell assays, respectively. Afterwards, effects of RUNX2 on of the activation of the PI3K/AKT and ERK pathways were explored by Western blot analysis. In addition, a PI3K/AKT pathway inhibitor LY294002 and an ERK inhibitor U0126 were applied to further verify the regulatory relationship between RUNX2 and the PI3K/AKT and ERK signaling pathways. Besides, the RUNX2 function on tumor formation in vivo was investigated by tumor xenograft experiment.. The result showed that RUNX2 was highly expressed in esophageal carcinoma tissues and cells. Knockdown of RUNX2 significantly inhibited TE-1 and EC-109 cell viability, repressed TE-1 cell migration and invasion, and increased TE-1 cell apoptosis. RUNX2 overexpression showed the opposite effects on HET-1A cells. Moreover, RUNX2-mediated TE-1 cell viability, migration and invasion were associated with the activation of the PI3K/AKT and ERK pathways. Besides, knockdown of RUNX2 markedly suppressed tumor formation in vivo.. Our results indicate that RUNX2 may play an oncogenic role in esophageal carcinoma by activating the PI3K/ AKT and ERK pathways. RUNX2 may serve as a potent target for the treatment of esophageal carcinoma.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromones; Core Binding Factor Alpha 1 Subunit; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Male; Mice; Mice, Nude; Middle Aged; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction

The membrane-cytoskeleton link organizer ezrin may be the most "dramatic" tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector.

Topics: Alternative Splicing; Binding Sites; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Codon, Initiator; Cytoskeletal Proteins; DNA; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; MAP Kinase Signaling System; Nitriles; Promoter Regions, Genetic; Tetradecanoylphorbol Acetate; Up-Regulation

Stomatin-like protein 2 (SLP-2), a member of the Stomatin superfamily, has been identified as an oncogenic-related protein and found to be up-regulated in multi-cancers. Nonetheless, the expression pattern and regulation of SLP-2 in human esophageal squamous cell carcinoma (ESCC) remain unexplored.. Immunohistochemistry and immunofluorescence staining analysis were performed to show SLP-2 expression and location. RNAi method was used to inhibit specific protein expression. Transwell assay was done to investigate cells invasive capability. RT-PCR and Western blot analysis were used to detect mRNA and protein expression levels.. Immunohistochemical analysis showed that up-regulation of SLP-2 was found in invasive front compared with cancer central tissue in ESCC. Inhibition of SLP-2 by SLP-2 siRNA can decrease ESCC cells invasive capability through MMP-2 dependent manner. Up-regulation of SLP-2 was effectively abrogated by the ERK1/2 inhibitors either PD98059 or U0126, but no effect was showed by the treatment of AKT inhibitors either LY294002 or MK-2206. So the regulation of SLP-2 was involved in activation of the MAPK/ERK pathway.. We found that PMA/EGF could induce the up-regulated expression of SLP-2 probably through activating ERK signalling. The current study suggests that SLP-2 may represent an important molecular hallmark that is clinically relevant to the invasion of ESCC.

Topics: Blood Proteins; Blotting, Western; Butadienes; Carcinoma, Squamous Cell; Esophageal Neoplasms; Flavonoids; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MAP Kinase Signaling System; Membrane Proteins; Neoplasm Invasiveness; Nitriles; RNA Interference

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.

Topics: Apoptosis; Blotting, Western; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phenotype; Phosphorylation; Real-Time Polymerase Chain Reaction; Up-Regulation

While there have been more and more studies concerning mitogen-activated protein kinases (MAPKs) signaling pathways, which control many cellular complex programmes, such as cell proliferation, differentiation, cell death and embryogenesis. However, few studies are carried out about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) in human esophageal cancer cell line. Therefore, in the present study, we investigated the expression and activation of ERK1/2 in human esophageal cancer cell line EC9706 and human normal esophageal epithelial cell line Heepic, which is as control. This study showed that ERK1/2 was transiently phosphorylated both in EC9706 and Heepic, the kinetics of which were slightly different. To further study the ERK/MAPK signaling pathway in EC9706 and Heepic cell line, U0126 a kind of specific inhibitor of MEK was used. This study showed that U0126 can block the phosphorylation of ERK1/2 in a short time, the complete inhibition concentration for EC9706 and Heepic cell line is 50 and 20 μM, respectively. Incidentally, to further investigate the different roles of ERK1 and ERK2, vector-based short hairpin interference vectors targeted on ERK1/2 was constructed. Moreover, the effective interference target sequence was screened out in a transient transfection manner. MTT experiment showed that ERK2 is more important than ERK1 in the proliferation of EC9706 cells.

Topics: Base Sequence; Butadienes; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Molecular Sequence Data; Nitriles; Phosphorylation; Signal Transduction

To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients.. The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas.. ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 µmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92 ± 3.49 in the ESCC tissues and 3.67 ± 7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P < 0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P > 0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87 ± 0.14 and 0.79 ± 0.10 in the ESCC tissues, and 1.10 ± 0.13 and 1.32 ± 0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P < 0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P > 0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P < 0.05).. ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.

Topics: Butadienes; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Line, Tumor; China; Enzyme Inhibitors; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; RNA, Messenger

The use of nonsteroidal anti-inflammatory drugs is associated with a lower risk for esophageal squamous cell carcinoma, in which overexpression of cyclooxygenase-2 (COX-2) is frequently reported. Prostaglandin E(2) (PGE(2)), a COX-2-derived eicosanoid, is implicated in the promotion of cancer growth. However, the precise role of PGE(2) in the disease development of esophageal squamous cell carcinoma remains elusive. In this study, we investigated the effect of PGE(2) on the proliferation of cultured esophageal squamous cell carcinoma cells (HKESC-1). Results showed that HKESC-1 cells expressed all four series of prostaglandin (EP) receptors, namely, EP1 to EP4 receptors. In this regard, PGE(2) and the EP2 receptor agonist (+/-)-15-deoxy-16S-hydroxy-17-cyclobutyl PGE(1) methyl ester (butaprost) markedly increased HKESC-1 cell proliferation. Moreover, the mitogenic effect of PGE(2) was significantly attenuated by RNA interference-mediated knockdown of the EP2 receptor, indicating that this receptor mediated the mitogenic effect of PGE(2). In this connection, PGE(2) and butaprost induced phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), whose down-regulation by RNA interference significantly attenuated PGE(2)-induced cell proliferation. In addition, PGE(2) and butaprost increased c-Fos expression and activator protein 1 (AP-1) transcriptional activity, which were abolished by the mitogen-activated protein kinase/Erk kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate (U0126). AP-1-binding inhibitor curcumin also partially reversed the mitogenic effect of PGE(2). Taken together, these data demonstrate for the first time that the EP2 receptor mediates the mitogenic effect of PGE(2) in esophageal squamous cell carcinoma via activation of the Erk/AP-1 pathway. This study supports the growth-promoting action of PGE(2) in esophageal squamous cell carcinoma and the potential application of EP2 receptor antagonists in the treatment of this disease.

Topics: Alprostadil; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Enzyme Activation; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Mitogens; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-fos; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Transcription Factor AP-1

Esophageal adenocarcinoma often arises from Barrett's esophagus. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) play critical roles in cell survival. We hypothesized that inhibition of these pathways in Barrett's adenocarcinoma would decrease cell proliferation and alter apoptosis in vitro.. Two Barrett's-associated adenocarcinoma cell lines, SEG-1 (wild-type p53) and BIC-1 (mutant p53), were treated with MAPK (U0126) and PI3K (LY294002) inhibitors at 20 microm concentrations. After 24 and 72 h, cell viability was measured by MTT assay. Apoptosis and necrosis were evaluated by the Annexin V-FITC assay. Statistical analysis was performed by ANOVA.. LY294002 and U0126 treatment produced significant reductions (range 15.7 to 62.0%, P < 0.05) in cellular proliferation at both 24 and 72 h in the SEG-1 cells. BIC-1 cell viability was reduced (39.3 to 56.4%, P < 0.05) at 72 h. Both early and late apoptotic activity were significantly increased (P < 0.05) in the SEG-1 cells using both inhibitors. Necrosis was significantly reduced (P < 0.05) using both inhibitors. No changes in either early or late apoptosis or necrosis were observed in the BIC-1 cells.. Herein, we report significant antiproliferative effects against Barrett's adenocarcinoma by MAPK and PI3K inhibition in vitro. Pro-apoptotic mechanisms prevail in the wild-type p53 cells. Further investigation is warranted to advance the clinical treatment of this devastating disease.

Topics: Adenocarcinoma; Apoptosis; Barrett Esophagus; Butadienes; Cell Division; Cell Line, Tumor; Cell Survival; Chromones; Enzyme Inhibitors; Esophageal Neoplasms; Humans; Mitogen-Activated Protein Kinases; Morpholines; Necrosis; Nitriles; Phosphoinositide-3 Kinase Inhibitors