u-0126 and Carcinoma--Squamous-Cell

Cancer is the most common cause of death worldwide with approximately one third of people being diagnosed with cancer in their lifetime. Pinostilbene hydrate (PSH) A methylated derivative of resveratrol Has been reported to possess antioxidative Cardioprotective and anticancer properties. However the antimetastatic effect of pinostilbene in oral squamous cell carcinoma (OSCC) remains unknown.. In this study We investigated the effect of PSH on antimetastatic activity and the relevant signaling pathways underlying mechanisms of SCC-9 SAS and HSC-3 oral cancer cell lines by MTT assay Wound healing Transwell assay Zymography and western blot analysis.. Our findings indicated that PSH inhibits migration and invasion ability by reducing the protein activity and expression of matrix metalloproteinases-2 (MMP-2) in all three cell lines. Moreover • The phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (p38) had significant inhibitory effects in the presence of PSH in the SCC9 and SAS cell lines. A combination of ERK1/2 and p38 inhibitors with PSH also reduced the migration and activity of MMP-2 in the SCC9 and SAS cell lines.. This study demonstrated that PSH suppresses MMP-2 enzymatic activity by downregulating the p38/ERK1/2 pathway and that it might be a promising agent for preventing OSCC cell metastasis.

Topics: Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Survival; Down-Regulation; Humans; Imidazoles; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Signal Transduction; Stilbenes

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

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

Galanin and its receptors, GALR1 and GALR2, are tumor suppressors and represent therapeutic targets in head and neck squamous cell carcinoma (HNSCC). In the present study, it was demonstrated that the re‑expression of GALR1 in GALR1 and GALR2‑negative HNSCC cells suppresses tumor cell proliferation. This is mediated via extracellular‑regulated protein kinase‑1/2 (ERK1/2)‑dependent effects on the cyclin‑dependent kinase inhibitors (CKI) and cyclin D1. In combination with galanin, GALR2 also suppressed proliferation by increasing CKI and decreasing cyclin D1 levels. In contrast to GALR1, overexpression of GALR2 also induced caspase‑3‑dependent apoptosis. It was identified that in GALR2‑transfected cells, galanin induced activation of ERK1/2 and suppressed cell proliferation. Galanin stimulation also decreased the expression of cyclin D1 and induced apoptotic DNA ladder formation in GALR2‑transfected cells. Pretreatment with the ERK1/2‑specific inhibitor U0126 and pertussis toxin prevented the suppression of cyclin D1 expression, however did not affect DNA ladder formation. In conclusion, GALR2 expression in the presence of galanin exerts antitumor effects via cell cycle arrest and apoptotic pathways, and reactivation of these pathways may have therapeutic benefits in HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Galanin; Gene Expression Regulation; Head and Neck Neoplasms; Humans; MAP Kinase Signaling System; Nitriles; Receptor, Galanin, Type 2; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway possesses confirmed oncogenic potential in oral squamous cell carcinoma (OSCC). Crosstalk with other molecular pathways contributes to STAT3 regulation in cancer. The effects of mitogen-activated protein kinases (MAPKs) and particularly extracellular signal-regulated kinase 1/2 (Erk1/2) on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examined the effects of Erk1/2 modulation on STAT3 signaling and cell growth in OSCC cells. Constitutive expression levels of phosphorylated (tyrosine and serine) and total STAT3, Erk1/2 and cyclin D1 were assessed in OSCC cell lines. Erk1/2 modulation was achieved by pharmacological agents; siRNA silencing against Erk1/2 was also performed. Cell proliferation and viability were assessed. Erk1/2 inhibition with either U0126 treatment or specific siRNA silencing resulted in decreases in p-ser STAT3 and cyclin D1 levels and increases in p-tyr STAT3 in OSCC cells. Moreover, Erk1/2 inhibition resulted in a dose-dependent reduction in OSCC cell growth and viability. Erk1/2 induction had the opposite effects. Taken together, these results are supportive of an active crosstalk between the oncogenic Erk1/2 and STAT3 pathways in OSCC, the significance of which requires further investigation.

Topics: Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Humans; MAP Kinase Signaling System; Mouth Neoplasms; Nitriles; Phosphorylation; RNA, Small Interfering; STAT3 Transcription Factor

Radioresistance limits the effectiveness of radiotherapy in head and neck squamous cell carcinoma. We previously demonstrated post-radiogenic mitogen-activated protein kinase (MAPK) pathway activation and vascular endothelial growth factor (VEGF) release resulting in reduced tumor cell response. Here, we examined the association of this mechanism with the induction of reactive oxygen species (ROS) under irradiation (IR).. Intracellular ROS after IR were measured. We modeled radiation-induced ROS by exposure of two SCC lines to H2 O2 and evaluated the impact of irradiation and ROS on ERK phosphorylation by Western blot, immunohistochemistry, and ELISA.. We found elevated pERK levels after treatment with IR and H2 O2 , which could be distinctly suppressed by U0126. Immunohistochemistry and ELISA revealed increased intracellular VEGF levels after H2 O2 application.. Our data show that irradiation-induced ROS activate the MAPK pathway and release of VEGF. As VEGF is known to be released after cellular distress resulting in cytoprotection, the described mechanism is potentially of importance for therapy success.

Topics: Blotting, Western; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytoprotection; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Humans; Hydrogen Peroxide; Immunohistochemistry; MAP Kinase Signaling System; Nitriles; Radiation Tolerance; Reactive Oxygen Species; Up-Regulation; Vascular Endothelial Growth Factor A

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

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

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

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

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

Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.. An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.. The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.. Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.

Topics: Antineoplastic Agents; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Chemoradiotherapy; Dasatinib; Drug Screening Assays, Antitumor; Head and Neck Neoplasms; Humans; Imidazoles; Isoxazoles; Leflunomide; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Radiation Tolerance; STAT5 Transcription Factor; STAT6 Transcription Factor; Thiazoles; Tumor Suppressor Proteins

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

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.

Topics: Antibodies, Monoclonal; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cysteine-Rich Protein 61; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha6beta1; Matrix Metalloproteinase 3; Mitogen-Activated Protein Kinases; Mouth Neoplasms; NF-kappa B; Nitriles; Phosphorylation; Promoter Regions, Genetic; Receptors, Vitronectin; Transcription Factor RelA

Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures.. Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation.. FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization.. Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy.

Topics: Butadienes; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Focal Adhesion Kinase 1; Head and Neck Neoplasms; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Targeted Therapy; Neoplasm Proteins; Nitriles; Paxillin; Phosphorylation; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Tumor Stem Cell Assay

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

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

Irradiation plays a pivotal role in head and neck squamous cell carcinoma (HNSCC) treatment. However, especially recurrent tumors frequently show increased radioresistance. We analyzed irradiation-stimulated mitogen-activated protein kinase (MAPK) signaling pathways to define cellular rescue mechanisms.. Irradiated HNSCC cells were screened for MAPK activation and results were confirmed and refined by functional analyses. Extracellular signal-regulated kinase (ERK) inhibitor U0126 application enabled us to specify postradiogenic cellular responses. Vascular endothelial growth factor (VEGF) levels were analyzed additionally.. We observed a pronounced and time-dependent ERK stimulation. Pathway inhibition resulted in decreased radioresistance. Likewise, we found a decrease of VEGF release after inhibitor treatment. ERK activation was confirmed in xenotransplants showing elevated postradiogenic phospho-ERK (pERK) and VEGF levels.. Our data give evidence for induction of ERK and successive VEGF release in HNSCC during radiotherapy, which might be partially explained by autoregulated cytoprotection maintained by pERK and potentially VEGF. In conclusion, targeting the ERK-VEGF axis might enhance the efficiency of radiotherapy.

Topics: Animals; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Head and Neck Neoplasms; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Nitriles; Radiotherapy Dosage; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A

Human papillomaviruses (HPVs) are a causative factor in over 90% of cervical and 25% of head and neck squamous cell carcinomas (HNSCCs). The C terminus of the high-risk HPV 16 E6 oncoprotein physically associates with and degrades a non-receptor protein tyrosine phosphatase (PTPN13), and PTPN13 loss synergizes with H-Ras(V12) or ErbB2 for invasive growth in vivo. Oral keratinocytes that have lost PTPN13 and express H-Ras(V12) or ErbB2 show enhanced Ras/RAF/MEK/Erk signaling. In co-transfection studies, wild-type PTPN13 inhibited Ras/RAF/MEK/Erk signaling in HEK 293 cells that overexpress ErbB2, EGFR or H-Ras(V12), whereas an enzymatically inactive PTPN13 did not. Twenty percent of HPV-negative HNSCCs had PTPN13 phosphatase mutations that did not inhibit Ras/RAF/MEK/Erk signaling. Inhibition of Ras/RAF/MEK/Erk signaling using MEK inhibitor U0126 blocked anchorage-independent growth in cells lacking PTPN13. These findings show that PTPN13 phosphatase activity has a physiologically significant role in regulating MAP kinase signaling.

Topics: Animals; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Human papillomavirus 16; Humans; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Nitriles; Oncogene Proteins, Viral; Papillomavirus Infections; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 13; Receptor, ErbB-2; Repressor Proteins

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

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

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Topics: Alkaloids; Amino Acid Sequence; Benzophenanthridines; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth Mucosa; Mouth Neoplasms; Nitriles; Phenanthridines; Protein Kinase C; Protein Kinase Inhibitors

In the United States, oral cancer accounts for more deaths annually than cervical cancer, leukemias, or Hodgkin's lymphoma. Studies have shown that aberrations of chromosome 18q develop with tumor progression and are associated with significantly decreased survival in head and neck cancer patients. The G-protein-coupled receptor, galanin receptor 1 (GALR1), maps to this region of chromosome 18q. Although the role of GALR1 has been well characterized in neuronal cells, little is known regarding this receptor in non-neuronal cells. In this study, the expression, mitogenic function, and signaling mechanism of GALR1 are investigated in normal and malignant oral epithelial cells. mRNA expression was determined via reverse transcriptase-PCR. Protein quantification was done via immunoblot analysis and enzyme-linked immunosorbent assay. For functional and signaling studies, an inhibitory antibody was generated to the N-terminal ligand binding domain of GALR1. GALR1 protein and mRNA expression and GAL secretion were detected at variable levels in immortalized human oral keratinocytes and human oropharyngeal squamous cell carcinoma cell lines. Upon competitive inhibition of GALR1, proliferation was up-regulated in immortalized and malignant keratinocytes. Furthermore, studies with the inhibitory antibody and U0126, the MAPK inhibitor, show that GALR1 inhibits proliferation in immortalized and malignant keratinocytes by inactivating the MAPK pathway. GALR1s inhibitory effects on proliferation in epithelial cells raises the possibility that inactivation or disregulation of this receptor can lead to uncontrolled proliferation and neoplastic transformation.

Topics: Animals; Antibodies; Antineoplastic Agents; Binding, Competitive; Butadienes; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; CHO Cells; Chromosomes, Human, Pair 18; Cricetinae; Culture Media, Conditioned; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Galanin; Humans; Immunoblotting; Keratinocytes; Ligands; MAP Kinase Signaling System; Models, Biological; Mouth Neoplasms; Nitriles; Protein Binding; Protein Structure, Tertiary; Receptor, Galanin, Type 1; Receptor, Galanin, Type 2; Receptor, Galanin, Type 3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Up-Regulation

Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features.. We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression.. Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects.. These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Nitriles; Osteopontin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Sialoglycoproteins; Signal Transduction; Survival Analysis; Up-Regulation

Interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) promote tumor angiogenesis, growth, and metastasis and are coexpressed by human head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers. The promoters of the IL-8 and VEGF genes contain different recognition sites for transcription factors nuclear factor (NF)-kappaB and activator protein-1 (AP-1), which we showed previously are coactivated in HNSCCs. NF-kappaB and AP-1 may be modulated by the inhibitor kappaB kinase (IKK) and mitogen-activated protein kinase (MAPK) signal pathways, but the contribution of these pathways to expression of IL-8 and VEGF and as potential targets for antiangiogenesis therapy in HNSCC is not known. In this study, we examined the effects of modulation of the MAPK and IKK pathways on expression of IL-8 and VEGF by UM-SCC-9 and UM-SCC-11B cell lines. Interruption of IKK-mediated activation of NF-kappaB by expression of an inhibitor kappaB alpha mutant (IkappaB alphaM) in UM-SCC-9 cells resulted in partial inhibition of expression of IL-8 but not VEGF. Analysis of possible alternative pathways for induction of these genes revealed activation of the MAPK extracellular signal-regulated kinase (ERK1/2) in cell lines UM-SCC-9 and UM-SCC-11B. Basal and tumor necrosis factor-alpha-inducible phosphorylation of ERK1/2 and secretion of IL-8 and VEGF could be specifically inhibited by a MEK inhibitor, U0126. Expression of IL-8 and VEGF in the cell lines was associated with coactivation of both NF-kappaB and AP-1, and U0126 inhibited both NF-kappaB and AP-1 reporter activity in UM-SCC-9 and UM-SCC-11B cells. The ERK pathway appears to contribute to expression of IL-8 and VEGF and transactivation of NF-kappaB as well as AP-1 in HNSCC. Combined inhibition of both MAPK and IKK pathways may be needed for suppression of the signal transduction mechanism(s) regulating VEGF and IL-8 secretion and angiogenesis by human HNSCC.

Topics: Butadienes; Carcinoma, Squamous Cell; Endothelial Growth Factors; Enzyme Inhibitors; Head and Neck Neoplasms; Humans; I-kappa B Kinase; Interleukin-8; Lymphokines; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth an

Topics: Antibodies; Breast Neoplasms; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Carcinoma, Squamous Cell; Cell Death; Cell Division; DNA, Antisense; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinases; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine