u-0126 and Adenocarcinoma

We previously reported that 37-kDa laminin receptor precursor involved in metastasis of lung adenocarcinoma cancer cells. In this study, we further revealed that hypoxia induced 37-kDa laminin receptor precursor expression and activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in lung adenocarcinoma cancer cells. In addition, we further demonstrated that the c-Jun N-terminal kinase inhibitor SP600125 and extracellular signal-regulated protein kinase inhibitor U0126 blocked the c-Jun activity and abolished hypoxia-induced 37-kDa laminin receptor precursor expression and promoter activity in a concentration-dependent manner. However, the p38 mitogen-activated protein kinase inhibitor did not affect 37-kDa laminin receptor precursor expression and c-Jun activity in response to hypoxia. Furthermore, downregulated c-Jun expression by short interfering RNA could also inhibit hypoxia-induced 37-kDa laminin receptor precursor expression and transcriptional activity. The inhibition of 37-kDa laminin receptor precursor expression by SP600125 and U0126 could be rescued by c-Jun overexpression. Studies using luciferase promoter constructs revealed a significant increase in the activity of promoter binding in the cells exposed to hypoxia, which was lost in the cells with mutation of the activator protein 1 binding site. Electrophoresis mobility shift assay and chromatin immunoprecipitation demonstrated a functional activator protein 1 binding site within 37-kDa laminin receptor precursor gene regulatory sequence located at -271 relative to the transcriptional initiation point. Hypoxia-induced invasion of A549 cells was inhibited by the pharmacologic inhibitors of c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated protein kinase (U0126) as well as 37-kDa laminin receptor precursor-specific siRNA or antibody. Our results suggest that hypoxia-elicited c-Jun/activator protein 1 regulates 37-kDa laminin receptor precursor expression, which modulates migration and invasion of lung adenocarcinoma cells.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Anthracenes; Butadienes; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Kinase 4; MAP Kinase Signaling System; Nitriles; Phosphorylation; Receptors, Laminin

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; B7-H1 Antigen; Butadienes; Cell Adhesion; Cell Movement; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutation; Nitriles; Phorbols; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factor AP-1; Transcription Initiation Site

Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Claudins; Fruit; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Promoter Regions, Genetic; Quercetin; RNA, Messenger; Up-Regulation; Vegetables

Growth and inflammatory factors are associated with poor prognosis in gastric adenocarcinoma (GA); however, the additive effects of growth and inflammatory factors in GA remain unclear. In this study, we investigated the ability of epidermal growth factor (EGF) and interleukin (IL-1β) to activate extracellular signal-regulated kinase (ERK)1/2 in GA cells, and correlated the relationships between their roles with the metastatic potential both in GA cells and GA tissues. The effects of EGF, IL-1β and EGF plus IL-1β in AGS and MKN-45 GA cells were examined using western blotting, Transwell migration and invasion assays, immunocytochemical staining and an activator protein (AP)-1 luciferase reporter gene assay, and was further characterized in GA tissues by immunohistochemistry. The results exhibited that EGF and IL-1β additively activated ERK1/2, increased migration and invasion than either EGF or IL-1β alone in AGS and MKN-45 cells. The mechanisms were involved in upregulating MMP-9 expression through increasing AP-1 transcriptional activity via ERK1/2 pathway; these effects were dose-dependently inhibited by silencing ERK1/2 or using U0126. In vivo data also confirmed that the overexpression of p-ERK1/2 in GA tissues correlated well with the EGF, IL-1β, EGF plus IL-1β, and was associated with metastasis, which was well correlation with the expression of MMP-9 and c-fos (AP-1). The results demonstrate that growth and inflammatory factors play an important role in metastasis of GA by additively activating ERK-1/2 and AP-1, and upregulating MMP-9. As both cytokines contribute to the migration and invasion of GA cells, EGF/IL-1β/ERK1/2 pathways may be key pathways closely associated with GA progression.

Topics: Adenocarcinoma; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Interleukin-1beta; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Nitriles; Phosphorylation; Stomach Neoplasms; Transcription Factor AP-1

Castration-resistant prostate cancer is an incurable heterogeneous disease that is characterized by a complex multistep process involving different cellular and biochemical changes brought on by genetic and epigenetic alterations. These changes lead to the activation or overexpression of key survival pathways that also serve as potential therapeutic targets. Despite promising preclinical results, molecular targeted therapies aimed at such signaling pathways have so far been dismal. In the present study, we used a PTEN-deficient mouse model of prostate cancer to show that plasticity in castration-resistant tumors promotes therapeutic escape. Unlike castration-naïve tumors which depend on androgen receptor and PI3K/AKT signal activation for growth and survival, castration-resistant tumors undergo phenotypic plasticity leading to increased intratumoral heterogeneity. These tumors attain highly heterogeneous phenotypes that are characterized by cancer cells relying on alternate signal transduction pathways for growth and survival, such as mitogen-activated protein kinase and janus kinase/signal transducer and activator of transcription, and losing their dependence on PI3K signaling. These features thus enabled castration-resistant tumors to become insensitive to the therapeutic effects of PI3K/AKT targeted therapy. Overall, our findings provide evidence that androgen deprivation drives phenotypic plasticity in prostate cancer cells and implicate it as a crucial contributor to therapeutic resistance in castration-resistant prostate cancer. Therefore, incorporating intratumoral heterogeneity in a dynamic tumor model as a part of preclinical efficacy determination could improve prediction for response and provide better rationale for the development of more effective therapies.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Butadienes; Carcinogenesis; Cell Proliferation; Combined Modality Therapy; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Everolimus; Humans; Male; Mice; Mice, Transgenic; Molecular Targeted Therapy; Nitriles; Orchiectomy; Phenotype; Prostate; Prostatic Neoplasms, Castration-Resistant; PTEN Phosphohydrolase; Receptors, Androgen; Sirolimus

To test the hypothesis that chemoresistance in pancreatic cancer is mediated via extracellular signal-regulated protein kinase (ERK) 1/2 overactivity.. The human pancreatic cancer cell lines BxPC3, PANC-1 and a stably gemcitabine-resistant subline, PANC1(GemRes), were treated with combinations of gemcitabine and the ERK1/2 inhibitor, U0126. Phosphorylated (p)ERK1/2 was examined by Western blotting; cell proliferation and apoptosis were quantified. A nude mouse xenograft model was established with each cell line, and the therapeutic efficacy of gemcitabine and U0126 alone or in combination was examined.. Gemcitabine treatment visibly increased pERK1/2 levels in BxPC-3 and PANC-1 cells. PANC-1(GemRes) constitutively produced high levels of pERK1/2. U0126 treatment reversed the gemcitabine-associated increase in cell proliferation and reduction in apoptosis, in all three cell lines. Combination treatment with U0126 and gemcitabine inhibited tumour growth and promoted apoptosis in xenograft tumours derived from all three cell lines.. ERK1/2 activity may protect pancreatic cancer cells from chemotherapy-induced apoptosis. The combined use of an ERK1/2 inhibitor (such as U0126) together with gemcitabine may result in synergistic therapeutic effects at tolerable gemcitabine doses.

Topics: Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Butadienes; Cell Line, Tumor; Deoxycytidine; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Gemcitabine; Humans; Inhibitory Concentration 50; Mice; Nitriles; Pancreatic Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Tumor Burden

Granulocyte colony-stimulating factor (G-CSF) affects granulopoiesis and is important for mobilizing neutrophils into blood circulation. Due to the hematopoietic properties of G-CSF, it has been widely used to clinically treat chemotherapy-induced neutropenia. However, G-CSF can promote tumors by inhibiting innate and adaptive immunity and enhancing angiogenesis and neoplastic growth. Most G-CSF-producing tumors are associated with a poor prognosis. This indicates that G-CSF promotes cancer progression. Thus, identifying regulatory molecules involved in tumor-derived G-CSF expression may provide therapeutic targets for cancer treatment. This study identified considerable G-CSF expression in malignant breast, lung and oral cancer cells. However, G-CSF expression was barely detectable in non-invasive cell lines. Expression of G-CSF mRNA and protein increased during exposure to tumor necrosis factor-α (TNF-α). Treatment with U0126 (a mitogen-activated protein kinase inhibitor) drastically reduced basal levels of G-CSF and TNF-α-induced G-CSF in aggressive cancer cells. This study also showed that knockdown of extracellular signal-regulated kinase (ERK) 2 by shRNA was necessary and sufficient to eliminate the expression of tumor-derived G-CSF. This did not apply to ERK1. Therefore, ERK2 (but not ERK1) is responsible for the transcriptional regulation of tumor-derived G-CSF. The results indicate the pharmaceutical value of specific ERK2 inhibitors in treating patients with G-CSF-producing tumors.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Breast Neoplasms; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasms; Nitriles; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Necrosis Factor-alpha

We have recently identified tumor necrosis factor (TNF)-α-induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr-669 and Ser-1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand-induced phosphorylation of serine and threonine residues in EGFR-overexpressing MDA-MB-468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr-669 and Ser-1046/1047. Inversely, constitutive tyrosine phosphorylation of the C-terminal domain, including Tyr-1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand-induced Thr-669 phosphorylation as well as Tyr-1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr-669 for Ala. In an asymmetric EGFR homodimer structure, one Thr-669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr-669 in an EGFR-ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK-mediated Thr-669 phosphorylation suppresses constitutive tyrosine phosphosphorylation in the homo- and heterodimer asymmetric conformations of the EGFR.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dimerization; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Humans; Ligands; MAP Kinase Signaling System; Neoplasm Proteins; Neuregulin-1; Nitriles; Phosphorylation; Phosphothreonine; Protein Conformation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Tumor Necrosis Factor-alpha

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin-biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Binding Sites; Butadienes; Cell Line, Tumor; Claudins; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Nitriles; Promoter Regions, Genetic; Protease Inhibitors; Protein Binding; Proto-Oncogene Proteins c-fos; Quinazolines; RNA, Messenger; RNA, Small Interfering; Transcription Factor AP-1; Tyrphostins

Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. This study aimed to explore the relationship between ERK, synergy/antagonism and cell cycle arrest in combination treatment.. A549 (mutant KRAS), H322 (wildtype KRAS) and siRNA-mediated KRAS knockdown A549 were treated with gemcitabine and/or the EGFR inhibitor AG1478 and analyzed with median effect analysis. Cell cycle distribution and ERK phosphorylation were assessed using flow cytometry and ELISA, respectively. Effect on cytotoxicity after ERK inhibition by U0126 was also assessed.. Cytotoxic interaction was dose dependent with antagonism at high dose AG1478. G1 arrest was observed with both high dose AG1478 and high dose gemcitabine and therefore was inconsistently associated with antagonism. Furthermore, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. ERK's effect in antagonism was further confirmed by using U0126. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment.. Our findings are consistent with a model in which ERK phosphorylation favors synergy and the outcome depends on the balance between gemcitabine-induced and AG1478-inhibited ERK phosphorylation. KRAS mutation confers resistance to AG1478 as well as combination treatment.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Deoxycytidine; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gemcitabine; Humans; Mutation; Nitriles; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; RNA, Small Interfering; Tyrphostins

The runt-domain transcription factor Runx3 plays crucial roles during development such as regulating gene expression. It has been shown that Runx3 is involved in neurogenesis, thymopoiesis and functions like a tumor suppressor. Runx3 null mouse die soon after birth as a result of multiple organ defects. Runx3 null mouse lung shows an abnormal phenotype and loss of Runx3 induced remodeling in the lung. Interestingly, lung adenocarcinoma is observed in Runx3 heterozygous mice at 18 months of age. During lung development various cellular and molecular events occur such as cell proliferation, cell death, differentiation and epithelial-mesenchymal transition (EMT). To understand the specific lethal events in Runx3 null mice, we examined cellular and molecular networks involved in EMT, and EMT inducers were quantified by RT-qPCR during lung development. Excessive EMT was observed in lungs at PN1 day in Runx3 null mice and PN18 months in Runx3 heterozygous mice. Pharmacologic inhibition of EMT was used to curb tumor progression. In this study, U0126 was injected to pregnant mouse for inhibition of pERK signaling. After U0126 treatment, life spans of newborn mice were increased and lung hyperplasia was partially rescued by down-regulated cell proliferation and EMT. Our data suggest that Runx3 is involved in crucial regulation of alveolar differentiation and tumor suppression in developing mouse lung.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Butadienes; Cell Differentiation; Cell Transformation, Neoplastic; Core Binding Factor Alpha 3 Subunit; eIF-2 Kinase; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Developmental; Lung; Lung Neoplasms; Mice; Mice, Knockout; Nitriles; Pregnancy; Pulmonary Alveoli; Signal Transduction

Hepatocyte growth factor (HGF) plays an important role in stimulating tumor invasion and metastasis of tumor cells. However, the exact mechanisms of HGF regulation of cell invasion are largely unknown. In this study, we demonstrated that the MAPK specific inhibitor U0126 prevented the HGF-induced invasion of SKOV-3 cells. HGF stimulation of cell invasion and enhancement of MMP-9 expression were partially suppressed by TSP-1 overexpression. Furthermore, the MAPK signaling pathway is predominantly responsible for HGF-induced TSP-1 downregulation. We conclude that HGF is a potential downregulator of TSP-1, through MAPK signaling pathways, leading to the induction of MMP-9 expression and subsequent invasion of SKOV-3 cells. This study enhances our understanding of the mechanisms of signaling transduction in the HGF-induced invasion and progression of ovarian cancer.

Topics: Adenocarcinoma; Butadienes; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; Nitriles; Ovarian Neoplasms; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Thrombospondin 1; Transfection

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17beta-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER alpha as compared to 17beta-estradiol and genistein. Despite poor binding to ER alpha, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER alpha at Ser(118). It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cyclic AMP; Drug Interactions; Estradiol; Estrogen Receptor alpha; Female; Genistein; Humans; Isoflavones; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Phytoestrogens; Signal Transduction

We previously reported that nicotine promoted gastric cancer cell growth via upregulation of cyclooxygenase 2 (COX-2). In the present study, we further investigated whether beta-adrenoceptors, protein kinase C (PKC), and extracellular signal-regulated kinase-1/2 (ERK1/2) were involved in the modulation of COX-2 expression and cell proliferation by nicotine in AGS, a human gastric adenocarcinoma cell line. Results showed that nicotine dose dependently increased the phosphorylation of EKR1/2 and the expression of AP-1 subunits c-fos and c-jun. In this connection, the ERK1/2 inhibitor U0126 abrogated the upregulation of AP-1 and COX-2 as well as cell proliferation induced by nicotine. Moreover, nicotine induced the translocation of PKC-betaI from cytosol to membrane and increased the total levels of PKC expression. Inhibition of PKC by staurosporine attenuated nicotine-induced ERK1/2 phosphorylation and COX-2 expression. Furthermore, atenolol and ICI 118,551, a beta1- and beta2-adrenoceptor antagonist, respectively, reversed the stimulatory action of nicotine on the expression of PKC, ERK1/2 phosphorylation, and COX-2 together with cell proliferation. Collectively, these results suggest that nicotine stimulates gastric cancer cell growth through the activation of beta-adrenoceptors and the downstream PKC-betaI/ERK1/2/COX-2 pathway.

Topics: Adenocarcinoma; Adrenergic beta-Antagonists; Atenolol; Butadienes; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Dose-Response Relationship, Drug; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Proteins; Mitogens; Nicotine; Nitriles; Phosphorylation; Propanolamines; Protein Kinase C; Protein Kinase C beta; Protein Kinase Inhibitors; Protein Transport; Receptors, Adrenergic, beta; Signal Transduction; Staurosporine; Stomach Neoplasms; Transcription Factor AP-1

The short fatty acid, butyrate, which is produced by intestinal anaerobic bacteria in the colon, has inhibitory activity on histone deacetylases (HDACs). Treatment of the human colon cancer cell line, LS174T, with 1-2 mM sodium butyrate stimulated MUC2 mucin production, as determined by histological PAS staining of carbohydrate chains of mucin, and confirmed at the protein and mRNA levels by immunoblotting with anti-MUC2 antibody and real-time RT-PCR, respectively. Increases in acetylated histone H3 in the LS174T cells treated with butyrate suggest inhibition of HDACs in these cells. Butyrate-stimulated MUC2 production in the LS174T cells was inhibited by the MEK inhibitor, U0126, implicating the involvement of extracellular signal-regulated kinase (ERK) cascades in this process. Proliferation of the LS174T cells was inhibited by butyrate treatment. Although apoptotic nuclear DNA fragmentation could not be detected, cell-cycle arrest at the G0/G1 phase in the butyrate-treated cells was demonstrated by flow cytometry. Thus butyrate, an HDAC inhibitor, inhibits proliferation of LS174T cells but stimulates MUC2 production in individual cells.

Topics: Adenocarcinoma; Butadienes; Butyrates; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Humans; Mitogen-Activated Protein Kinase Kinases; Mucin-2; Mucins; Nitriles

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

To clarify the involvement of matrix metalloproteinase-7 (MMP-7) in cell dissociation and the subsequent invasion of pancreatic cancer cells.. Western blotting, in vitro invasion assay, immunocytochemistry, and immunohistochemistry were performed in pancreatic cancer cell lines or pancreatic cancer tissue.. The active form of the MMP-7 protein was expressed exclusively in the conditioned medium of dissociated (PC-1.0 and AsPC-1) pancreatic cancer cells, whereas proMMP-7 protein was only detected in the conditioned medium of non-dissociated (PC-1 and Capan-2) cells. Both intracellular and conditioned medium localised MMP-7 was greatly reduced by treatment with the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the mitogen activated protein kinase kinase (MEK) inhibitor U0126 in pancreatic cancer cells. MMP-7 treatment significantly induced the disruption of tight junction (TJ) structures and subsequent cell dissociation, and activation of the EGFR mediated MEK- ERK (extracellular signal regulated protein kinase) signalling pathway in the non-dissociated pancreatic cancer cells. Moreover, the strong in vitro invasiveness of dissociated cells was inhibited by AG1478 and U0126 treatment, whereas the weak invasiveness of non-dissociated cells was apparently induced by MMP-7 treatment. In addition, MMP-7 expression was stronger at the invasive front than at the centre of human pancreatic tumours.. MMP-7 is involved in cell dissociation and the subsequent invasion of pancreatic cancer cells. It induces the disruption of TJ structures and forms a positive feedback loop with activation of the EGFR mediated MEK-ERK signalling pathway.

Topics: Adenocarcinoma; Adult; Aged; Blotting, Western; Butadienes; Culture Media, Conditioned; Down-Regulation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Male; Matrix Metalloproteinase 7; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Nitriles; Pancreatic Neoplasms; Quinazolines; Signal Transduction; Tight Junctions; Tumor Cells, Cultured; Tyrphostins

Cyclooxygenase-2 (COX-2) and ERK-MAPK mitogenic signaling pathways are important in human hepatocellular carcinoma. We investigated the effect of COX-2 inhibition on ERK-MAPK signaling and the effect of combining MEK (MAPK kinase) and COX-2 inhibitors in human hepatocellular carcinoma in vitro. COX and ERK expression were determined by immunoblot in HepG2 and Hep3B cells. COX-2 and MEK activity were determined by prostaglandin E(2) assay and phosphospecific immunoblot, respectively. Cell growth was determined by cell proliferation and cell counts. Apoptosis was determined by DNA fragmentation enzyme-linked immunosorbent assay and flow cytometry. Cell cycle was determined by flow cytometry. HepG2 and Hep3B cells do not express COX-1 or COX-2. Correspondingly, basal and agonist (arachidonic acid, lipopolysaccharide)-stimulated COX-2 activity is undetectable. Treatment of HepG2 and Hep3B cells with NS398 resulted in an increase in ERK1/2 phosphorylation (MEK activity) in a concentration-dependent fashion (NS398, 1 to 100 micromol/L). Treatment with the COX-2 inhibitor NS398 in the presence of U0126 (MEK inhibitor) effectively suppressed ERK1/2 phosphorylation as determined by phosphospecific ERK1/2 immunoblot. Total ERK1/2 and COX-2 were unchanged with NS398 and U0126 treatments. In HepG2 cells, NS398 (1 to 100 micromol/L) decreased apoptosis as determined by DNA fragmentation enzyme-linked immunosorbent assay. Relative apoptosis was increased with U0126 alone or in combination with NS398 (9 to 10 times the control value), eliminating the anti-apoptotic effect of NS398. In Hep3B cells, apoptosis was unchanged with NS398 (1 to 50 micromol/L) or U0126 (1 to 10 micromol/L) alone. The combination of NS398 and U0126 in Hep3B cells resulted in a synergistic increase in apoptosis (10 times the control value). Relative apoptosis in both cell lines strongly correlated with changes in the expression of the antiapoptotic protein Bcl-xL. Cellular growth was assessed by colorimetric proliferation assay and cell counts. HepG2 and Hep3B cells had concentration-dependent inhibition of cell growth with NS398 or U0126 treatment alone. The combination of NS398 and U0126 resulted in complementary inhibitory effects on growth. Growth inhibitory effects in HepG2 and Hep3B cells with combination treatment appear to be, in part, secondary to the induction of G(0)/G(1) and G(2)/M cell cycle arrest, respectively, as determined by flow cytometry. Despite differential signaling in HepG

Topics: Adenocarcinoma; Apoptosis; Butadienes; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Drug Synergism; Enzyme Inhibitors; Humans; Isoenzymes; Liver Neoplasms; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Nitrobenzenes; Pancreatic Neoplasms; Prostaglandin-Endoperoxide Synthases; Signal Transduction; Sulfonamides

Interactions between cancer cells and their microenvironment are critical for the development and progression of solid tumors. This study is the first to examine the role of all members of the ErbB tyrosine kinase receptors (epidermal growth factor receptor [EGFR], ErbB-2, ErbB-3, or ErbB-4), expressed singly or as paired receptor combinations, in the regulation of angiogenesis both in vitro and in vivo. Comparison of all receptor combinations reveals that EGFR/ErbB-2 and ErbB-2/ErbB-3 heterodimers are the most potent inducers of vascular endothelial growth factor (VEGF) mRNA expression compared with EGFR/ErbB-3, EGFR/ErbB-4, ErbB-2/ErbB-4, and ErbB-3/ErbB-4. Immunohistochemistry of tumor xenografts overexpressing these heterodimers shows increased VEGF expression and remarkably enhanced vascularity. Enhanced VEGF expression is associated with increased VEGF transcription. Deletional analysis reveals that ErbB-mediated transcriptional up-regulation of VEGF involves a hypoxia-inducible factor 1-independent responsive region located between nucleotides -88 to -66 of the VEGF promoter. Mutational analysis reveals that the Sp-1 and AP-2 transcription factor binding elements within this region are required for up-regulation of VEGF by heregulin beta1 and that this up-regulation is dependent on the activity of extracellular signal-related protein kinases. These results emphasize the biological implications of cell signaling diversity among members of the ErbB receptor family in regulation of the tumor microenvironment.

Topics: Adenocarcinoma; Animals; Butadienes; Cell Line; Dimerization; DNA-Binding Proteins; Endothelial Growth Factors; Enzyme Inhibitors; ErbB Receptors; Genes, Reporter; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Kruppel-Like Transcription Factors; Lymphokines; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Neuregulin-1; Nitriles; Promoter Regions, Genetic; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Recombinant Fusion Proteins; Signal Transduction; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transplantation, Heterologous; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Estrogen acts to promote DNA synthesis in the MCF-7 human breast cancer cell line via its interaction with high levels of estrogen receptor. The primary mode of estrogen action has been considered to be through transcriptional activation of genes containing estrogen response elements, including the immediate early genes c-myc and fos. Recent reports have indicated that estrogen, acting through the estrogen receptor, is capable of inducing the mitogen-activated protein kinase (MAPK) cytoplasmic signaling cascade. In this study, specific small molecule inhibitors of MAPK and phosphatidylinositol 3-kinase activity were used to determine the influence of these cascades on estrogen-mediated mitogenesis. Phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, as well as inhibitors of MAPK kinase-1, PD098059 and U0126, decreased the fraction of cells entering DNA synthesis after treatment with 17beta-estradiol. These compounds did not inhibit expression of myc or fos. However, the drugs did prevent the accumulation of cyclin D1 and hyperphosphorylated retinoblastoma protein, indicating that the block occurred at, or prior to, this point in the cell cycle. Although these compounds were effective in preventing estrogen-mediated mitogenesis, the downstream kinases extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, and protein kinase B were not activated over basal levels by estrogen treatment. These studies suggest that estrogen initiates mitogenesis by inducing the transcription of immediate early genes, but cytoplasmic signaling pathways play an important role in the control of subsequent events in the cell cycle.

Topics: Adenocarcinoma; Androstadienes; Breast Neoplasms; Butadienes; Chromones; Culture Media, Serum-Free; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Depression, Chemical; DNA Replication; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Flavonoids; Fulvestrant; Humans; Isoquinolines; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mitosis; Morpholines; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Sulfonamides; Wortmannin