u-0126 and Carcinoma--Non-Small-Cell-Lung

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

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

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

Protein kinase D1 (PKD1) is increasingly implicated in multiple biological and molecular events that regulate the proliferation or invasiveness in several cancers. However, little is known about the expression and functions of PKD1 in non-small cell lung cancer (NSCLC). In the present study, 34 pairs of human NSCLC and matched normal bronchiolar epitheliums were enrolled and evaluated for PKD1 expression by quantitative real-time PCR. We showed that PKD1 was downregulated in 26 of 34 cancer tissues in comparison with matched normal epitheliums. Moreover, patients with venous invasion or lymph node metastasis showed significant lower expression of PKD1. Exposure of NSCLC A549 and H520 cells to the PKD family inhibitor kb NB 142-70(Kb), at concentrations that inhibited PKD1 activation, strikingly potentiated S6K1 phosphorylation at Thr(389) and S6 phosphorylation at Ser(235/236) in response to phorbol ester (PMA). Knockdown of PKD1 with siRNAs strikingly enhanced S6K1 phosphorylation whereas constitutively active PKD1 resulted in the S6K1 activity inhibition. Furthermore, the PI3K inhibitors LY294002, BKM120 and MEK inhibitors U0126, PD0325901 blocked the enhanced S6K1 activity induced by Kb. Collectively, our results identify decreased expression of the PKD1 as a marker for NSCLC and the loss of PKD1 expression increases the malignant potential of NSCLC cells. This may be due to the function of PKD1 as a negative regulator of mTORC1-S6K1. Our results suggest that re-expression or activation of PKD1 might serve as a potential therapeutic target for NSCLC treatment.

Topics: Aminopyridines; Benzamides; Blotting, Western; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromones; Diphenylamine; Enzyme Activation; Humans; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Nitriles; Phorbol Esters; Real-Time Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases; TRPP Cation Channels

KRAS mutations are found in 15-25 % of patients with lung adenocarcinoma, and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway, which are mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK) and PI3K pathway (PI3K-AKT-mTOR). Here, we examined the inhibition of both these pathways alone and in combination and analyzed the anti-proliferative and apoptotic events in KRAS mutant NSCLC cell lines, A549 and Calu-1. Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), and RAD001 (mTOR inhibitor) for 24 and 48 h. The expression levels of p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27(kip1), BAX, BIM, and GAPDH were detected by western blot after 6 and 24 h treatment. Although PI3K/mTOR inhibition is more effective in cytotoxicity in A549 and Calu-1 cells, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Our data show that combined targeting of MEK and PI3K-AKT with mTOR is a better option than single agents alone for KRAS mutant NSCLC, thus opening the possibility of a beneficial treatment strategy in the future.

Topics: Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Chromones; Elafin; Everolimus; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Morpholines; Mutation; Nitriles; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; TOR Serine-Threonine Kinases

Sorafenib is an inhibitor of a number of intracellular signaling kinases with antiproliferative, anti‑angiogenic and pro‑apoptotic effects in tumor cells. Sorafenib has been used in the therapy of advanced renal cell carcinoma. In the present study, using two human non‑small cell lung cancer (NSCLC)cell lines, A549 and NCI‑H1975, the effects of sorafenib on proliferation, apoptosis and intracellular signaling were systematically characterized. The results revealed that at a low concentration (5 µM) and early time point (6 h), sorafenib is capable of significantly stimulating proliferation of A549 cells, but not NCI‑H1975 cells. In addition, the comparison of the two cell lines revealed different cell cycle redistribution and apoptotic susceptibility to sorafenib at this concentration and time point. Western blot analysis revealed that sorafenib upregulated the expression of cyclin D1 and cyclin‑dependent kinase 2 and downregulated the expression of BAX at this specific point. Furthermore, sorafenib was confirmed to regulate the expression of cyclin D1 and apoptosis‑associated proteins through the regulation of extracellular signal‑regulated kinase 1/2 phosphorylation in A549 cells. These findings suggest that, although sorafenib has the potential for use in the treatment of renal cell carcinoma, this compound may also activate NSCLC cells at a specific time point.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; Niacinamide; Nitriles; Phenylurea Compounds; Phosphorylation; Signal Transduction; Sorafenib

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

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

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

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

β-Carboline alkaloids are naturally occurring plant substances that have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Recently, we have demonstrated that harmol, a β-carboline alkaloid, induces apoptosis by caspase-8 activation independently from Fas/Fas ligand interaction in human non-small cell lung cancer (NSCLC) H596 cells. Here, we found that harmol induces autophagy and cell death in human NSCLC A549 cells. Although harmol induced cell death in A549 cells in a significant dose- and time-dependent manner, it did not induce caspase-3, caspase-8, or caspase-9 activity. Furthermore, cleavage of poly-(ADP-ribose)-polymerase was not induced in A549 cells by harmol treatment. Autophagy, but not apoptosis, was detected by electron microscopy in A549 cells treated with 70 µM harmol. Pretreatment of A549 cells with 3-methyladenine, an autophagy inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of LC3, both suppressed harmol-induced cell death. These suggest that the induction of autophagy by harmol precedes cell death. The cytotoxicity of some anticancer agents is reportedly linked to autophagy induction. The 2 major autophagy regulatory pathways are the Akt/mammalian target of rapamycin (mTOR) pathway and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Although harmol treatment showed no effect on the Akt/mTOR pathway, it transiently activated the ERK1/2 pathway. However, inhibition of the ERK1/2 pathway using the mitogen-activated protein kinase (MEK)/ERK inhibitor U0126 partially suppressed autophagy. Therefore, although activation of the ERK1/2 pathway might be related to harmol-induced autophagy, another major pathway may also be involved in A549 cells.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Harmine; Humans; Microscopy, Electron; Nitriles; Phytotherapy; Plant Extracts; RNA, Small Interfering

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

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

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

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

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