u-0126 and Lung-Neoplasms

Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust.. Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method.. Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased.. Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.. 【中文题目：瘦素/ERK信号在云锡矿粉诱导
大鼠II型肺泡上皮细胞转化中的作用】 【中文摘要：背景与目的 目前，云南个旧锡矿有大量矿工从事开采工作，这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关，大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡II型上皮细胞（immortalized rat alveolar cells type II, RLE-6TN）恶性转化过程中，瘦素（leptin）及其介导的细胞外调节蛋白激酶（extracellular regulated protein kinase, ERK）信号通路所起的作用。方法 采用200 μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代，建立毒染细胞模型，命名为R₂₀₀细胞，正常培养组命名为R细胞，通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）抑制剂（U0126）对R₂₀₀细胞的最佳作用浓度。自第20代起，将R组、R₂₀₀组细胞分别与leptin及MEK抑制剂U0126共培养，对各组细胞的形态改变进行观察，并利用苏木素-伊红（hematoxylin-eosin, HE）染色技术鉴别第40代细胞的形态学差异，通过刀豆凝集素A（concanavalin A, ConA）及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。结果 R组和R₂₀₀组细胞均表达leptin受体（OB-R）。与R₂₀₀组比较，当leptin浓度达100 ng/mL时，其促增殖效应最为显著，30 μmol/L U0126可抑制毒染细胞R₂₀₀增殖，与对照组相比具有统计学差异（P<0.05）。自第25代起，leptin诱导的R₂₀₀组（R₂₀₀L组）细胞形态发生变化，至第30代出现恶性转化，至第40代时恶性转化特征明显；而R₂₀₀组细胞及U0126诱导的R₂₀₀组（R₂₀₀LU组）细胞则在第40代时才出现恶性转化特征。R₂₀₀L组细胞凝集速度较R₂₀₀LU组快，其余各组细胞P30出现凝集，且随ConA浓度增加，细胞凝集速度加快。R₂₀₀L组细胞自P40可见克隆形成，克隆形成率为2.25‰±0.5‰，R₂₀₀LU组及R₂₀₀组未见克隆集落。R₂₀₀L组细胞pERK表达增强；加入U0126阻断后，R₂₀₀L组细胞pERK磷酸化水平降低。结论 Leptin可以促进云锡矿粉毒染肺上皮细胞的恶性转化，ERK信号通路可能是其促进云锡矿粉引发的肺泡II型上皮细胞转化的重要途径。
】 【中文关键词：瘦素；肺泡II型上皮细胞；转化；ERK信号通路】.

Topics: Alveolar Epithelial Cells; Animals; China; Dust; Epithelial Cells; Leptin; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Rats; Receptors, Leptin; Signal Transduction; Tin

Brain metastasis (BM) is associated with poor prognosis in patients with advanced non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutation reportedly enhances the development of BM. However, the exact mechanism of how EGFR-mutant NSCLC contributes to BM remains unknown. Herein, we found the protein WNT5A, was significantly downregulated in BM tissues and EGFR-mutant samples. In addition, the overexpression of WNT5A inhibited the growth, migration, and invasion of EGFR-mutant cells in vitro and retarded tumor growth and metastasis in vivo compared with the EGFR wide-type cells. We demonstrated a molecular mechanism whereby WNT5A be negatively regulated by transcription factor E2F1, and ERK1/2 inhibitor (U0126) suppressed E2F1's regulation of WNT5A expression in EGFR-mutant cells. Furthermore, WNT5A inhibited β-catenin activity and the transcriptional levels of its downstream genes in cancer progression. Our research revealed the role of WNT5A in NSCLC BM with EGFR mutation, and proved that E2F1-mediated repression of WNT5A was dependent on the ERK1/2 pathway, supporting the notion that targeting the ERK1/2-E2F1-WNT5A pathway could be an effective strategy for treating BM in EGFR-mutant NSCLC.

Topics: Animals; beta Catenin; Brain Neoplasms; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Down-Regulation; E2F1 Transcription Factor; ErbB Receptors; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Nude; Mutation; Neoplasm Staging; Nitriles; RNA Interference; RNA, Small Interfering; Wnt-5a Protein

Topics: Acute Kidney Injury; Animals; Apoptosis; Blood Urea Nitrogen; Butadienes; Cell Proliferation; Cisplatin; Kidney; Lipocalin-2; Lung Neoplasms; MAP Kinase Signaling System; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; Neoplasm Proteins; Nitriles; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Tumor Burden

Lung cancer (LC) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Using bioinformatics analysis and immunohistochemical of lung carcinoma tissues, we show that TRIM59 as a critical oncoprotein relating to LC proliferation and metastasis. In this study, high TRIM59 expression was significantly correlated with lymph node metastasis, distant metastasis, and tumour stage. Furthermore, up-regulation of TRIM59 expression correlated with poorer outcomes in LC patients. Mechanistically, TRIM59 play a key role in promoting LC growth and metastasis through regulation of extracellular-signal regulated protein kinase (ERK) signalling pathway and epithelial-to-mesenchymal transition (EMT)-markers, as validated by loss-of-function studies. In-depth bioinformatics analysis showed that there is preliminary evidence of co-expression of TRIM59 and cyclin dependent kinase 6 (CDK6) in LC. Notably, CDK6 expression significantly decreased when TRIM59 was knocked down in the LC cells. In contrast, exogenous up-regulation of TRIM59 expression also induced significant increases in the expression of CDK6. Moreover, the expression of CDK6 was also inhibited by the ERK signalling inhibitor, U0126. The results of both loss- and gain-of-function studies showed that TRIM59 could regulate the expression of CDK6. Collectively, these data provide evidence that TRIM59 is involved in lung carcinoma growth and progression possibly through the induction of CDK6 expression and EMT process by activation of ERK pathway.

Topics: Biomarkers, Tumor; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 6; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis; Nitriles; Signal Transduction; Tripartite Motif Proteins

Lung cancer, especially the non-small-cell lung cancer, is a highly aggressive vascular cancer with excessively activated signaling pathways. Tumor-associated calcium signal transducer 2, also known as trop2, was identified to be correlated with tumor proliferation and invasion of non-small-cell lung cancer; however, the biological role of trop2 in neovascularization of non-small-cell lung cancer remained elusive. In this study, we first verified that trop2 was overexpressed in non-small-cell lung cancer tissues as well as cell lines and that the increased expression of trop2 promoted non-small-cell lung cancer cell proliferation and invasion. Then, we expanded the biological role of trop2 by in vitro and in vivo angiogenesis assay. The tubular formation analysis revealed that trop2 promoted non-small-cell lung cancer angiogenesis in vitro, and the immunohistochemistry staining of vascular markers (CD31 and CD34) provided evidences that trop2 promoted in vivo neovascularization. The results of polymerase chain reaction array revealed that trop2 promoted the expression level of two well-known angiogenesis factors MMP13 and PECAM1. By screening the trop2-related signaling pathways, we observed that excessive angiogenesis was correlated with activation of ERK1/2 signaling pathway, and ERK1/2 inhibitor (U0126) could suppress the tubular formation ability induced by trop2 expression. These results suggested that trop2 facilitated neovascularization of non-small-cell lung cancer via activating ERK1/2 signaling pathway. Targeting trop2 might provide novel anti-angiogenesis strategy for non-small-cell lung cancer treatment.

Topics: A549 Cells; Animals; Antigens, Neoplasm; Butadienes; Calcium; Carcinoma, Non-Small-Cell Lung; Cell Adhesion Molecules; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 13; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neovascularization, Pathologic; Nitriles; Platelet Endothelial Cell Adhesion Molecule-1; Polymerase Chain Reaction; Protein Structure, Tertiary

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

Lung cancer is the most common cause of cancer‑associated mortality worldwide, and glucosamine has the potential to exhibit antitumor activity. To reveal its anti‑lung cancer mechanism, the present study investigated the effect of glucosamine on the transcriptional activity of forkhead box O (FOXO)1 and FOXO3, and associated signal transduction pathways in A549 cells. An MTT assay was performed to investigate cell viability and immunoblotting was performed to detect protein levels of FOXO1/3, phosphorylated (p)‑FOXO1/3, AKT, p‑AKT, extracellular signal‑regulated kinase (ERK) and p‑ERK, and the levels of β‑O‑linked N‑acetylglucosamine (O‑GlcNAc)‑modified FOXO1 protein. Immunoprecipitation was performed to purify O‑GlcNAc‑modified protein prior to immunoblotting. Glucosamine inhibited FOXO1‑ and FOXO3‑specific amino acid phosphorylation, which was correlated with its translocation from the nucleus to cytoplasm, indicating a possible anti‑lung cancer mechanism of glucosamine. The present study also examined the phosphoinositide 3‑kinase (PI3K)/AKT and mitogen‑activated protein kinase (MAPK)/ERK pathways, which induce FOXO1‑ and FOXO3‑specific site phosphorylation. The data showed that glucosamine suppressed the translocation of FOXO from the cytoplasm to the nucleus via glucosamine‑induced O‑GlcNAc modification. These observations suggested that glucosamine modulated A549 cell proliferation, possibly via O‑GlcNAc modification‑induced downregulation of the PI3K/AKT and MAPK/ERK pathways and their downstream signaling molecules, FOXO1 and FOXO3.

Topics: A549 Cells; Antineoplastic Agents; Butadienes; Cell Proliferation; Chromones; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Forkhead Transcription Factors; Glucosamine; Humans; Lung Neoplasms; Morpholines; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-akt

Oleanolic acid (OA) is a natural triterpenoid that is widely distributed in edible and medicinal plants. OA exerts anti-tumor activity on a wide range of cancer cells primarily through inducing apoptosis. Dysregulated ERK signaling is closely complicated in the biology of cancer, such as metastasis, proliferation, and survival, and it can be activated by various stimuli. In this study, we found that OA induced the activation of ERK in cancer cells. ERK activation compromised the apoptosis induced by OA. Blocking ERK activation by U0126 or siRNAs was able to potentiate the pro-apoptotic activity of OA on cancer cells. OA was shown to promote ERK-dependent Nrf2 expression in cancer cells, and in turn, Nrf2 expression was able to suppress OA-induced ROS generation. Blockade of Nrf2 expression was able to increase ROS levels and apoptotic death in cancer cells. In conclusion, we provided evidences that ERK activation is a mechanism underlying the resistance of cancer cells to OA-induced apoptosis and targeting ERK is a promising strategy to enhance the anti-tumor efficacy of OA.

Topics: Animals; Apoptosis; Blotting, Western; Butadienes; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; NF-E2-Related Factor 2; Nitriles; Oleanolic Acid; Pancreatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Beauvericin (BEA) is a cyclic hexadepsipeptide that derives from Codyceps cicadae. Our previous study results indicated that the cytotoxic effects of BEA on human A549 lung cancer cells BEA occur through an apoptotic pathway, which involves the up-regulation of cytochrome c release from mitochondria, upregulation of caspase 3 activity, and cellular and morphological changes. In this study, we identified that the mitogen-activated protein kinase (MAPK) inhibitor U0126 inhibits the cytotoxic effects of BEA on A549 cells. After exposing human A549 cells to 10 μM BEA, we observed a significant and dose-dependent increase in the percentage of hypoploid (sub-G1) phase cells in the A549 population. Following the pretreatment of the A549 cells with 25 μM U0126, the distribution of A549 cells in the sub-G1 phase decreased significantly. The BEA treatment resulted in a significant increase apoptosis in A549 cells by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, the MEK1/2 (mitogen-activated protein kinase kinase)-ERK42/44 (extracellular signal-regulated kinases)-90RSK (ribosomal s6 kinase) signaling pathway was activated in BEA-induced apoptotic A549 cells. Furthermore, treatment with MEK1/2 inhibitor U0126 was capable to attenuate the BEA induced typical apoptotic morphological change, apoptotic cells, and MEK1/2-ERK42/44-90RSK signaling pathway. These results suggested that MEK1/2-ERK42/44-90RSK signaling pathway may play a important role in BEA-induced apoptosis in human NSCLC A549 cancer cells.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Survival; Depsipeptides; Dose-Response Relationship, Drug; G1 Phase Cell Cycle Checkpoints; Lung Neoplasms; Mitogen-Activated Protein Kinases; Nitriles; Protein Kinase Inhibitors; Signal Transduction

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

Almost all small-cell lung cancers (SCLC) and carcinoid tumors express neuroendocrine differentiation (NED), and 10% to 20% of non-small-cell lung cancers (NSCLC) are associated with NED. Although distinct clinical features and histology of SCLC and carcinoid tumors are well recognized, the clinical significance and the molecular basis of NED in NSCLC remain unclear.. To explore the potential molecular pathway involved in NED of NSCLC and its clinical relevance, we conducted investigations using an NSCLC cell line (NCI-H157) as a NED induction model, and explored the potential intracellular signal transduction pathways involved in NED of NSCLC. We confirmed our findings using activators versus inhibitors to these signal transduction pathways in vitro. We also performed immunohistochemical stains of phospho-Erk1/2 of lung cancer specimens known to have NED and explored its clinical relevance.. We discovered that NED of NSCLC was associated with the activation of Erk1/2-mitogen-activated protein kinases (MAPK) signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb) and inhibitors (siRNA-Erk1/2 and U0126) to the Erk1/2-MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2-MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2-MAP-kinase pathway revealed a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors.. To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Topics: Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Line, Tumor; Humans; Lead; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Neurosecretory Systems; Nitriles; Phosphopyruvate Hydratase; Phosphorylation; Proto-Oncogene Proteins c-akt

Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-κB signaling pathway effects on muscles is beneficial in cancer-induced cachexia.

Topics: Animals; Autophagy; Boronic Acids; Bortezomib; Butadienes; Cachexia; Cell Line, Tumor; Inflammation; Lung Neoplasms; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle Proteins; Muscle Weakness; Muscle, Skeletal; NF-kappa B; Nitriles; Oxidation-Reduction; Proteolysis; Pyrazines; Signal Transduction; Sulfasalazine; Tyrosine; Weight Gain

Combination chemotherapy with multiple drugs has been widely applied to cancer treatment owing to enhanced efficacy and reduced drug resistance. For drug combination experiment analysis, response surface modeling has been commonly adopted. In this paper, we introduce a Hill-based global response surface model and provide an application of the model to a 512-run drug combination experiment with three chemicals, namely AG490, U0126, and indirubin-3  ' -monoxime (I-3-M), on lung cancer cells. The results demonstrate generally improved goodness of fit of our model from the traditional polynomial model, as well as the original Hill model on the basis of fixed-ratio drug combinations. We identify different dose-effect patterns between normal and cancer cells on the basis of our model, which indicates the potential effectiveness of the drug combination in cancer treatment. Meanwhile, drug interactions are analyzed both qualitatively and quantitatively. The distinct interaction patterns between U0126 and I-3-M on two types of cells uncovered by the model could be a further indicator of the efficacy of the drug combination.

Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; Butadienes; Cell Line, Tumor; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Humans; Indoles; Lung Neoplasms; Models, Statistical; Nitriles; Oximes; Tyrphostins

High expression levels of cyclooxygenase-2 (COX-2) contribute a strong proliferative ability to human lung cancer cells, and this function is link to the epidermal growth factor receptor (EGFR) pathway, which was mediated by extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NFκB). In this study, scutellarein, a flavonoid compound, was screened for proliferation inhibition at different concentrations (0, 5, 25 and 50 μM) at 24 h or 48 h in human lung cancer cell line A549. Results showed that A549 cell proliferation was inhibited by 50 μM scutellarein treatment in 24 h and 48 h of treatment. The expression levels of phosphorylated EGFR, phosphorylated ERK, phosphorylated NFκB and COX-2 were reduced in a dose-dependent manner after 24 h scutellarein treatments at different concentrations. Further, ERK inhibitor U0126 and NFκB inhibitor MG132 also inhibited A549 cell proliferation similar to 50 κM scutellarein treatment from 24 h to 48 h. The experimental results showed that scutellarein could inhibit proliferation of the human lung cancer cell line A549 through ERK and NFκB mediated by the EGFR pathway.

Topics: Antineoplastic Agents; Apigenin; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Leupeptins; Lung Neoplasms; MAP Kinase Signaling System; NF-kappa B; Nitriles

FOXM1, a member of the Forkhead transcriptional family, plays an important role in the EMT process, and transforming growth factor-β1 (TGF-β1) has been identified as the most potent factor that can independently induce EMT in various types of cancer cells. Here we examine the important role of FOXM1 in TGF-β1-induced EMT and investigate the mechanism underlying the relationship between TGF-β1 and FOXM1. Lentivirus-mediated transfection was used to stably upregulate the expression of FOXM1, and a small interfering RNA (siRNA) was introduced to silence the expression of FOXM1. Transwell and wound-healing assays were then performed to assess the invasion and motility potential of non-small cell lung cancer (NSCLC) cells. The NSCLC cell lines exhibited EMT characteristics, including an elongated fibroblastoid shape, induced expression of EMT marker proteins, and increased migratory and invasive potential after induction with TGF-β1. The overexpression of FOXM1 enhanced TGF-β1-induced EMT in NSCLC cells. Knockdown of FOXM1 reversed TGF-β1-induced EMT in NSCLC cell lines but had no effect on the phosphorylation level of ERK. Additionally, U0126, an ERK signaling inhibitor, exerted a reversible effect on TGF-β1-induced EMT and inhibited FOXM1 expression. FOXM1 regulated by the ERK pathway can mediate TGF-β1-induced EMT in NSCLC and is a potential target for the treatment of NSCLC.

Topics: Biomarkers, Tumor; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Molecular Targeted Therapy; Neoplasm Invasiveness; Nitriles; RNA, Small Interfering; Transforming Growth Factor beta1; Transgenes; Wound Healing

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

To investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer.. Normal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected.. The miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells.. The activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.

Topics: Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Humans; Lung; Lung Neoplasms; MAP Kinase Signaling System; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Quinazolines; Tyrphostins

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Topics: Aged; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mitogen-Activated Protein Kinases; Mutation; Nitriles; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyridazines; ras Proteins; RNA Interference

The emergence of radioresistance is a significant issue in the treatment of squamous cell carcinoma. We recently demonstrated that post-radiogenic extracellular signal-regulated kinase (ERK) signaling might decrease radiosensitivity in this cancer type. To further elucidate how tumor-organizing cell types respond to irradiation and ERK pathway inhibition, we analyzed one oral squamous cell carcinoma and one lung cancer cell line (HNSCCUM-02T, A549), fibroblasts (NIH3T3), primary normal and cancer-associated fibroblasts (CAFs) in vitro. Irradiated cells treated with mitogen-activated protein kinase (MAPK) inhibitor U0126 were screened for pERK levels. Post-radiogenic cellular responses were functionally analyzed by proliferation and colony assays. We found analogous pERK expression, proliferation and survival of tumor and normal fibroblast cells. CAFs did not show any response to treatment. We hypothesized that radiation and MAPK inhibition have no dose-limiting effect on tumor-surrounding normal tissue. As CAFs are considered to influence the radioresponse of the entire tumor, but are not affected by treatment themselves, potential CAF-mediated tumor protection should be considered in further studies.

Topics: Animals; Butadienes; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mouth Neoplasms; NIH 3T3 Cells; Nitriles; Protein Kinase Inhibitors; Radiation Dosage; Radiation Tolerance; Tumor Stem Cell Assay

The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.

Topics: Aged; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Interleukin-8; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mutation; Nitriles; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Phenotype; Pyridines; ras Proteins; RNA Interference; Smoking

Lung cancer is a malignant disease with poor outcome, which has led to a search for new therapeutics. The PI3K/Akt/mTOR and Ras/raf/Erk pathways are key regulators of tumor growth and survival. In the present study, their roles were evaluated by MTT assay, flow cytometry and Western blotting in lung cancer cells. We found that a high efficacy of antitumor activity was shown with GDC-0941 treatment in two gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines, A549 and H460. In addition, H460 cells with activating mutations of PIK3CA were relatively more sensitive to GDC-0941 than A549 cells with wild-type PIK3CA. Furthermore, GDC-0941 was highly efficacious in combination with U0126 in inducing cell growth inhibition, G0-G1 arrest and cell apoptosis. These antitumor activities of combined treatment may be attributed to the alterations of G0-G1 phase regulators, apoptosis-related proteins and eukaryotic translation initiation factor 4B (eIF4B), induced by concomitant blockade of the PI3K/Akt/mTOR and Ras/raf/Erk pathways. In conclusion, this study suggests that multi‑targeted intervention is the most effective treatment for tumors. Additionally, the blockade of PI3K, mTOR and Erk with GDC-0941 and MEK inhibitors shows promise for treating gefitinib-resistant NSCLC.

Topics: Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Synergism; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Humans; Indazoles; Lung Neoplasms; Mitogen-Activated Protein Kinases; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases

Estrogen has been reported to promote an increased susceptibility to lung cancer development. This study focusses on the role of cytoplasmic estrogen receptor β (c-ERβ) in NSCLC.. NSCLC (n = 162) cases were analyzed using immunohistochemistry (IHC) for c-ERβ expression and its association with clinicopathological variables. Significance of c-ERβ expression was further examined using in vitro studies in NSCLC cell lines.. Among ERβ and aromatase positive NSCLC females, c-ERβ was significantly associated with greater tumor diameter and tended to be associated with worse overall survival. A549 and LCAM1 cells expressed aromatase, as well as c-ERβ and nuclear ERβ (n-ERβ). U0126 (MAPK/extracellular-signal-regulated kinase (ERK) inhibitor) abrogated MAPK phosphorylation, caused by estradiol via c-ERβ, more effectively than ICI 182780 (ER blocker) in either cell line. However, ICI 182780 completely abrogated the estrogen responsive elements (ERE)-luciferase activity caused by estradiol. Combination therapy with ICI 182780 and U0126 turned out to be far more effective than either treatment alone in either A549 or LCAM1 cells.. The results indicated that ERβ may contribute to NSCLC via non-genomic action of estrogen through its cytoplasmic form, in addition to the genomic actions via n-ERβ. These actions of estrogen in NSCLCs may be abrogated by combination therapy with ICI 182780 and U0126.

Topics: Aromatase; Biomarkers, Tumor; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytoplasm; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Fulvestrant; Humans; Immunohistochemistry; Lung Neoplasms; Nitriles

Thymosin beta-4 (Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)-GSK-3α (inactive), β-catenin, E-cadherin and E-cadherin:β-catenin complex was relatively higher, p-GSK-3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

Topics: Animals; beta Catenin; Butadienes; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nitriles; Peptides; Phosphorylation; RNA Interference; RNA, Small Interfering; Stomach Neoplasms; Thymosin; Wnt Signaling Pathway

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

Metastasis remains the primary cause of lung cancer. The molecules involved in metastasis may be candidates for new targets in the therapy of lung cancer. The MEK/ERK signaling pathway has been highlighted in a number of studies on invasiveness and metastasis. In this paper, we show that the MEK inhibitor U0126 induces flattened morphology, remodels the actin-based cytoskeleton, and potently inhibits chemotaxis and Matrigel invasion in the human lung cancer A549 cell line. Furthermore, downregulation of ERK by small interfering RNA significantly inhibits the invasion of A549 cells and induces stress fiber formation. Taken together, our findings provide the first evidence that the inhibition of invasion of lung cancer A549 cells by inhibiting MEK/ERK signaling activity is associated with remodeling of the actin cytoskeleton, suggesting a novel link between MEK/ERK signaling-mediated cell invasion and the actin-based cytoskeleton.

Topics: Actin Cytoskeleton; Butadienes; Cell Movement; Humans; Lung Neoplasms; MAP Kinase Kinase Kinases; Neoplasm Invasiveness; Nitriles; Protein Kinase Inhibitors; RNA Interference

In this study, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in a platelet-activating factor (PAF)-induced experimental pulmonary tumor metastasis model. An adenovirus carrying PTEN cDNA (Ad-PTEN) reversed PAF-induced increase in phosphorylation of AKT as well as pulmonary metastasis of B16F10. PAF-induced pulmonary metastasis was inhibited by MAPK inhibitors, but not by PI3K inhibitor. Ad-PTEN abrogated PAF-induced phosphorylation of MAPKs. These data indicate PTEN/MAPK pathways play a key role in PAF-induced tumor metastasis.

Topics: Androstadienes; Animals; Anthracenes; Butadienes; Cell Line; Humans; Imidazoles; Lung Neoplasms; MAP Kinase Signaling System; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet Activating Factor; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyridines; Wortmannin

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

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. This study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured by using the 3-dimensional (3D) organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands, EGF, TGF-α, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MAP/ERK kinase (MEK) inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion by using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we showed that bronchial hyperplasia can be modeled in vitro by using primary NHTBE cells maintained in a 3D organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role.

Topics: Bronchi; Butadienes; Cell Line, Tumor; Cyclin D1; Enzyme Inhibitors; ErbB Receptors; Erlotinib Hydrochloride; Humans; Hyperplasia; Luciferases; Lung Neoplasms; MAP Kinase Kinase Kinases; Models, Biological; Nitriles; Organ Culture Techniques; Quinazolines; RNA, Small Interfering

Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII). However, little is known about the mechanism of down-regulation of TβRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-β-induced tumor suppressor function. Here we show that HDIs restore TβRII expression and that DNA hypermethylation has no effect on TβRII promoter activity in lung cancer cell lines. TGF-β-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TβRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TβRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TβRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TβRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TβRII in lung cancer and that TGF-β tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TβRII expression.

Topics: Animals; Blotting, Western; Butadienes; CCAAT-Binding Factor; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Nitriles; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous

To investigate the effects of gefitinib (EGFR-TKI), LY294002 (PI3K inhibitor) and U0126 (MEK inhibitor) on proliferation and apoptosis in five non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/AB2, H1975, H1299 and A549).. The inhibitory rates of cells were tested by MTT and apoptosis was detected through flow cytometry when treated with gefitinib, LY294002 and U0126.. The sensitivity to gefitinib was different in different cell lines, which was associated with EGFR mutation type. The cells with EGFR mutation were more sensitive than those with EGFR wild-type, except PC9/AB2 cells. LY294002 and U0126 can inhibit cell proliferation and promote apoptosis in all five cell lines. The sensitivity to gefitinib was restored partially in the resistant cell lines by combining gefitinib with LY294002 or U0126. The effects on cell proliferation and apoptosis were stronger in cells with EGFR mutation when PI3K/AKT pathway was blocked, however, for cells with EGFR wild-type, the effects were stronger when the MEK pathway was blocked. When the PI3K and MEK pathways were blocked together, proliferation inhibition and apoptosis level in NSCLC cells was similar to that in cells treated with EGFR TKI. There were some differences according to EGFR mutation type, suggesting that EGFR mutations may result in alterations of downstream signaling pathways.. The sensitivity of gefitinib resistant cell lines can be restored partially when the two downstream signaling pathways are blocked. However, these cells were still drug resistant, suggesting that the activation of PI3K and MEK pathways is not the only mechanism of EGFR-resistance.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Chromones; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Gefitinib; Humans; Lung Neoplasms; MAP Kinase Kinase Kinases; Morpholines; Mutation; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants; it exhibits an anticancer effect on many malignancies. The most important chemotherapeutic agent for patients with advanced non-small cell lung cancer (NSCLC) is a platinum-containing compound such as cisplatin or carboplatin. The molecular mechanism underlying decreased NSCLC cell viability after treatment with emodin and cisplatin is unclear. Therefore, the aim of this study was to assess the cytotoxic effect of combined emodin and cisplatin on NSCLC cell lines and to clarify underlying molecular mechanisms. Exposure of human NSCLC cells to emodin decreased cisplatin-elicited ERK1/2 activation and ERCC1 protein induction by increasing instability of ERCC1 protein. Cisplatin alone did not affect expression of ERCC1 mRNA. However, emodin alone or combined with cisplatin significantly decreased expression of ERCC1 mRNA levels. Enhancement of ERK1/2 activation by transfection with constitutively active MKK1/2 (MKK1/2-CA) vector increased ERCC1 protein levels and protein stability, as well as increasing viability of NSCLC cells treated with emodin and cisplatin. In contrast, blocking ERK1/2 activation by U0126 (an MKK1/2 inhibitor) decreased cisplatin-elicited ERCC1 expression and enhanced cisplatin-induced cytotoxicity. Depletion of endogenous ERCC1 expression by si-ERCC1 RNA transfection significantly enhanced cisplatin's cytotoxic effect. In conclusion, ERCC1 protein protects NSCLC cells from synergistic cytotoxicity induced by emodin and platinum agents. Further investigation of combined emodin and cisplatin may lead to novel therapy in the future for NSCLC through down-regulating expression of ERCC1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Cisplatin; DNA-Binding Proteins; Drug Synergism; Emodin; Endonucleases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; RNA, Small Interfering

Most non-small cell lung cancers (NSCLC) display elevated expression of epidermal growth factor receptor (EGFR), but response to EGFR kinase inhibitors is predominantly limited to NSCLC harboring EGFR-activating mutations. These mutations are associated with increased activity of survival pathways, including phosphatidylinositol 3-kinase/AKT and signal transducer and activator of transcription 3/5. We report that EGFR-activating mutations also surprisingly lead to decreased ability to activate extracellular signal-regulated kinase (ERK) compared with wild-type EGFR. In NSCLC cells and mouse embryonic fibroblasts expressing mutant EGFR, this effect on ERK correlates with decreased EGFR internalization and reduced phosphorylation of SHP2, a tyrosine phosphatase required for the full activation of ERK. We further show that ERK activation levels affect cellular response to gefitinib. NSCLC cells with EGFR mutation display reduced gefitinib sensitivity when ERK activation is augmented by expression of constitutively active mutants of mitogen-activated protein kinase/ERK kinase (MEK). Conversely, in a NSCLC cell line expressing wild-type EGFR, gefitinib treatment along with or following MEK inhibition increases death response compared with treatment with gefitinib alone. Our results show that EGFR-activating mutations may promote some survival pathways but simultaneously impair others. This multivariate alteration of the network governing cellular response to gefitinib, which we term "oncogene imbalance," portends a potentially broader ability to treat gefitinib-resistant NSCLC.

Topics: Animals; Antineoplastic Agents; Butadienes; Carcinoma, Non-Small-Cell Lung; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gefitinib; HeLa Cells; Humans; Lung Neoplasms; Mice; Mutation; Nitriles; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Quinazolines

The inflammatory cytokines tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) stimulate production of the inflammatory mediators prostaglandin E₂ (PGEγ), prostacyclin (PGIγ), and nitric oxide (NO) in cultured lung epithelial cells. Pretreatment of these cells with the selective MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase 1/2) inhibitor U0126 blocked ERK1/2 activation and inhibited cytokine-induced production of these inflammatory mediators. Primary bronchiolar epithelial Clara cells treated with TNFα and IFNγ also produced increased PGE₂, PGI₂, and NO, and PG and NO production was decreased by MEK inhibition. U0126 differentially affected cyclooxygenase (COX)-1, COX-2, and inducible NO synthase (iNOS) expression in cell lines, however, suggesting that MEK1/2 regulates prostanoid and NO production by means other than inducing their biosynthetic enzymes. Functionally, inhibition of MEK1/2 caused G1 cell cycle arrest and decreased cyclin D1 expression, but these effects were not related to decreased prostanoid production. These results indicate separate proinflammatory and proliferative roles for ERK1/2 in lung epithelial cells. During lung tumor formation in vivo, ERK1/2 phosphorylation increased as lung tumors progressed. Since tumor-derived cells were more sensitive than nontumorigenic cells to the antiproliferative effects of U0126, MEK1/2 inhibition may serve as an attractive chemotherapeutic target.

Topics: Animals; Apoptosis; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Interferon-gamma; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Nitric Oxide; Nitriles; Phosphorylation; Prostaglandins; Respiratory Mucosa; Tumor Necrosis Factor-alpha

Epidermal growth factor receptor (EGFR) inhibitors are highly effective in treating non-small cell lung cancers (NSCLC) expressing activated EGFR, particularly those harboring EGFR mutations.  However, most patients who benefit from EGFR inhibitors achieve only partial responses or stable disease, facilitating the emergence of resistance.  Thus, progression-free survival advantages in responding patients are modest.  Combination therapy, preferably using agents with synergistic activity, could both improve responses and reduce acquired resistance rates.   We hypothesized that combining MEK inhibitors with EGFR inhibitors could result in such a benefit.  The MAPK pathway lies downstream of EGFR and transduces both proliferative and survival signals in a variety of cancer types.  Inhibitors of this pathway are currently in clinical trials, but little evidence exists supporting the use of these agents as monotherapy in EGFR-dependent non-small cell lung cancer.   In this study, we find EGFR-dependent NSCLC cell lines are moderately sensitive to loss of ERK1/2 activity, either by small molecule inhibition or by siRNA knockdown.  The consequence of inhibition is dependent upon the trophic content of the culture media, primarily anti-proliferative in serum-rich conditions and pro-apoptotic in serum-poor conditions. However, when ERK inhibition combined with EGFR inhibitors, cytotoxic synergy was observed for all EGFR-dependent cell lines tested in serum-containing media.  Enhanced cytotoxicity is demonstrated in cell lines with and without EGFR mutations, including those harboring the T790M escape mutation.  These findings support future clinical studies that combine EGFR- and MEK1/2-targeted agents to investigate whether improved outcomes can be achieved in clinically screened EGFR-dependent NSCLC.

Topics: Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Enzyme Activation; ErbB Receptors; Gene Silencing; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Nitriles; Protein Kinase Inhibitors

Cell migration is a critical step in cancer cell invasion. Recent studies have implicated the importance of the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cell migration. However, the mechanism associated with ERK-regulated cell migration is poorly understood. Using a panel of breast cancer cell lines, we detected an excellent correlation between ERK activity and cell migration. Interestingly, we noticed that a 48-hour treatment with U0126 [specific mitogen-activated protein/ERK kinase (MEK)-1/2 inhibitor] was needed to significantly inhibit breast cancer cell migration, whereas this inhibitor blocked ERK activity within 1 hour. This observation suggests that ERK-dependent gene expression, rather than direct ERK signaling, is essential for cell migration. With further study, we found that ERK activity promoted the expression of the activator protein-1 (AP1) components Fra-1 and c-Jun, both of which were necessary for cell migration. Combination of U0126 treatment and Fra-1/c-Jun knockdown did not yield further reduction in cell migration than either alone, indicating that ERKs and Fra-1/c-Jun act by the same mechanism to facilitate cell migration. In an attempt to investigate the role of Fra-1/c-Jun in cell migration, we found that the ERK-Fra-1/c-Jun axis regulated slug expression in an AP1-dependent manner. Moreover, the occurrence of U0126-induced migratory inhibition coincided with slug reduction, and silencing slug expression abrogated breast cancer cell migration. These results suggest an association between ERK-regulated cell migration and slug expression. Indeed, cell migration was not significantly inhibited by U0126 treatment or Fra-1/c-Jun silencing in cells expressing slug transgene. Our study suggests that the ERK pathway regulates breast cancer cell migration by maintaining slug expression.

Topics: Animals; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Neoplasm Invasiveness; Nitriles; Oligonucleotide Array Sequence Analysis; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors

Histone deacetylase 6 (HDAC6) is a multifunctional, cytosolic protein deacetylase that primarily acts on alpha-tubulin. Here we report that stable knockdown of HDAC6 expression causes a decrease in the steady-state level of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor alpha, in A549 lung cancer cells. The decreased levels of in EGFR in HDAC6-knockdown cells, which correlated with increased acetylation of microtubules, were due to increased turnover of EGFR protein. Despite the decrease in EGFR levels, A549 cells lacking functional HDAC6 appeared to grow normally, probably due to increased expression of extracellular signal-regulated kinases 1 and 2. Indeed, HDAC6-knockdown cells were more sensitive than control cells to the MEK inhibitor U0126. These results suggest that HDAC6 inhibitors combined with inhibitors of growth factor signaling may be useful as cancer therapy.

Topics: Acetylation; Butadienes; Cell Line, Tumor; Cell Proliferation; Down-Regulation; ErbB Receptors; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Lung Neoplasms; Microtubules; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Protein Kinase Inhibitors; Receptor, Platelet-Derived Growth Factor alpha

Cisplatin is widely used for the treatment of solid tumors, including small cell lung cancers, but its success is often compromised due to relapse and resistance to further treatment. p70 ribosomal S6 kinase (p70S6K) has been shown to be upregulated in lung cancer cells. In the present study, we investigated whether the p70S6K pathway contributes to cisplatin resistance in human small cell lung cancer H69 cells. The levels of phosphorylated p70S6K and its downstream target S6 but not total p70S6K or S6 were elevated in the H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells. Cisplatin treatment resulted in the activation of p70S6K and downregulation of p70S6K was associated with cisplatin-induced PARP cleavage. While the ability of cisplatin to induce apoptosis was attenuated in H69/CP cells, inhibition of p70S6K by rapamycin enhanced cisplatin-induced apoptosis in these cells as evident by the increase in cisplatin-induced poly(ADP-ribose) polymerase (PARP) cleavage. The phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 alone induced PARP cleavage and further augmented cisplatin-induced PARP cleavage. In contrast, inhibition of extracellular signal-regulated kinase (ERK) by U0126 attenuated cisplatin-induced PARP cleavage. Both rapamycin and Ly294002 enhanced cisplatin-induced acti-vation of ERK1/2. Taken together, these results suggest that activation of p70S6K contributes to cisplatin resistance in small cell lung cancer H69 cells, and inhibition/downregulation of p70S6K as well as activation of ERK1/2 could circumvent cisplatin resistance.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Small Cell; Cell Line, Tumor; Chromones; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.

Topics: Amino Acid Substitution; Animals; Butadienes; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Nitriles; Proto-Oncogene Proteins B-raf; RNA, Neoplasm; RNA, Small Interfering; Signal Transduction

Silibinin, isolated from Silybum marianum, has been known for its hepatoprotective properties and recent studies have revealed its antiproliferative and apoptotic effects on several cancer cells. An inhibitory effect of silibinin on tumor invasion and matrix metalloproteinase-2 (MMP-2) and urokinasetype plasminogen activator (u-PA) activities in culture medium has been observed in our previous study and the impacts of silibinin on enzyme activities of MMPs, u-PA, mitogen-activated protein kinase (MAPK) and Akt in A549 cells were continued to explore in this study. Our results showed that silibinin exerted an inhibitory effect on the phosphorylation of Akt, as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are the members of the MAPK family involved in the up-regulation of MMPs or u-PA, while no effects on the activities of p38(MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase were observed. A treatment with silibinin to A549 cells also led to a dose-dependent inhibition on the activation of NF-kappaB, c-Jun and c-Fos. Additionally, the treatment of inhibitors specific for MEK (U0126) or PI3K (LY294002) to A549 cells could result in a reduced expression of MMP-2 and u-PA concomitantly with a marked inhibition on cell invasion. These findings suggested that the inhibition on MMP-2 and u-PA expression by silibinin may be through a suppression on ERK1/2 or Akt phosphorylation, which in turn led to the reduced invasiness of the cancer cells.

Topics: Anticarcinogenic Agents; Butadienes; Cell Line, Tumor; Cell Movement; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Lung Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Plant Extracts; Silybin; Silybum marianum; Silymarin; Urokinase-Type Plasminogen Activator

To study the expression of proline-rich Akt-substrate PRAS40 in the cell survival pathway and tumor progression.. The effects of three key kinase inhibitors on PRAS40 activity in the cell survival pathway, serum withdrawal, H(2)O(2) and overexpression of Akt were tested. The expression of PRAS40, Akt, Raf and 14-3-3 in normal cells and cancer cell lines was determined by Western blot.. The PI3K inhibitors wortmannin and Ly294002, but not rapamycin, completely inhibited the phosphorylation of Akt and PRAS40. The phosphorylation level of Akt decreased after serum withdrawal and treatment with the MEK inhibitor Uo126, but increased after treatment with H(2)O(2) at low concentration, whereas none of these treatments changed PRAS40 activity. 14-3-3 is a PRAS40 binding protein, and the expression of 14-3-3, like that of PRAS40, was higher in HeLa cells than in HEK293 cells; PRAS40 had a stronger phosphorylation activity in A549 and HeLa cancer cells than in HEK293 normal cells. In the breast cancer model (MCF10A/MCF7) and lung cancer model (BEAS/H1198/H1170) we also found the same result: PRAS40 was constitutively active in H1198/H1170 and MCF7 pre-malignant and malignant cancer cells, but weakly expressed in MCF10A and BEAS normal cell. We also discussed PRAS40 activity in other NSCLC cell lines.. The PI3K-Akt survival pathway is the main pathway that PRAS40 is involved in; PRAS40 is a substrate for Akt, but can also be activated by an Akt-independent mechanisms. PRAS40 activation is an early event during breast and lung carcinogenesis.

Topics: 14-3-3 Proteins; Adaptor Proteins, Signal Transducing; Breast Neoplasms; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydrogen Peroxide; Lung Neoplasms; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Transfection

We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.

Topics: Blotting, Northern; Blotting, Western; Butadienes; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Line, Tumor; Doxorubicin; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lung Neoplasms; Lymphoma; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 8; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly Adenosine Diphosphate Ribose; Pyridines; Urokinase-Type Plasminogen Activator

The effects of Dox (Dox), paclitaxel (Taxol), and serum starvation on the regulation of XIAP (X-linked inhibitor of apoptosis), Bcl-2 phosphorylation, and apoptosis were evaluated in human H460 non-small cell lung cancer cells. Protein kinases that responded to these treatments as prosurvival elements in signal transduction were identified by simultaneously screening phosphorylation of protein kinases in H460 cells cultured in serum-free medium or treated with Dox. We demonstrated that Dox and Taxol induced apoptosis through down-regulation of XIAP and phosphorylation of Bcl-2 in a concentration-dependent manner without changing expression of Bcl-xL in H460 cells. These effects were paralleled by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase protein. We identified that serum starvation and Dox reduced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK), protein kinase C (PKC) alpha/beta and c-Jun NH(2)-terminal kinase. The MEK-specific inhibitor U0126 or PKC inhibitor staurosporine (STP) also down-regulated XIAP expression and induced apoptosis. Thus, our data suggest that apoptosis and down-regulation of XIAP induced by Dox exposure or serum starvation may be mediated through inactivation of the MEK/ERK and PKCalpha/beta pathways. In support of this we demonstrated that the cytotoxic effects of Dox when combined with U0126 or STP were enhanced, i.e., synergistic cytotoxic activities were demonstrated. The synergistic interaction of U0126 or STP with Dox was sequence- and concentration-dependent.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Butadienes; Carcinoma, Non-Small-Cell Lung; Caspases; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Paclitaxel; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase C; Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Staurosporine; Tumor Cells, Cultured; X-Linked Inhibitor of Apoptosis Protein

A combination of paclitaxel (Taxol) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK/Erk) inhibitor represents a rational new approach to chemotherapy. We performed Affymetrix microarray analysis to understand the global effects of this combination in lung carcinoma. Genes involved in cell cycle control, apoptosis, adhesion, proliferation, invasion, and metastasis were modulated. We observed similar patterns of gene modulation in ovarian and melanoma cell lines, indicating the general applicability of these findings. Functional genomic analysis identified two genes as new targets of drug-induced tumor apoptosis. The MGSA/Gro1 gene, important in melanoma growth, was induced by paclitaxel and reduced by MEK inhibition. Blockage of paclitaxel-induced melanoma growth stimulatory activity significantly reduced melanoma growth. Additionally, the expression of topoisomerase III beta, which exhibited a clear pattern of gene reduction by a combination of the two drugs, was significantly increased (5.7-fold) in primary lung cancers but not adjacent tissues. These findings provide potential new biomarkers and gene targets for the development of improved cancer treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Dactinomycin; Enzyme Inhibitors; Female; Gene Expression Profiling; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Paclitaxel

The growing knowledge of the tight connection between apoptosis and cancer has lead to an explosion of research revolving around apoptotic induction with chemotherapeutic agents and small molecule inhibitors. The chemotherapeutic agent paclitaxel (Taxol) activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and, combined with MEK inhibition, synergistically enhances apoptosis. Here we implement a proteomic approach using two-dimensional gels coupled with mass spectrometry to identify proteins altered with this coordinated combination treatment. We found that the combined treatment of paclitaxel and MEK inhibitor uniquely altered the proteins RS/DJ-1 (RNA-binding regulatory subunit/DJ-1 PARK7) and RhoGDIalpha (Rho GDP-dissociation inhibitor alpha). Functional proteomic analysis by exogenous expression or short interfering RNA targeting confirmed a role in survival and apoptosis for these proteins. Analysis of primary lung tumors with matched adjacent normal tissue confirmed RS/DJ-1 overexpression in non-small cell lung carcinoma. This study shows the power of proteomic profiling coupled with functional analysis for the discovery of novel molecular targets and potential cancer cell-specific biomarkers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Enzyme Inhibitors; Guanine Nucleotide Dissociation Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; MAP Kinase Kinase Kinase 1; Nitriles; Oncogene Proteins; Paclitaxel; Protein Deglycase DJ-1; Protein Serine-Threonine Kinases; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; rho-Specific Guanine Nucleotide Dissociation Inhibitors