epidermal-growth-factor and Mouth-Neoplasms

Topics: Animals; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Herpes Simplex; Humans; Male; Mouth Neoplasms; Oncogenes; Papillomaviridae; Tumor Virus Infections

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Topics: Carcinoma, Squamous Cell; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Mouth Neoplasms; Proto-Oncogene Proteins c-cbl; Ubiquitin-Protein Ligases

Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. Although a variety of endocytic pathways are reportedly involved in the cellular uptake of exosomes, detailed mechanisms remain unknown. The present study demonstrated that treatment with recombinant epidermal growth factor (EGF) time- and dose-dependently promoted cellular uptake of oral squamous cell carcinoma (OSCC) cell-derived exosomes into OSCC cells themselves. Conversely, EGF receptor (EGFR) knockdown and treatment with EGFR inhibitors, including erlotinib and cetuximab, abrogated OSCC cell uptake of exosomes. The macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked the effects of active EGF/EGFR signaling on uptake of OSCC cell-derived exosomes. These EGFR inhibitors also suppressed OSCC cell-derived exosome-induced proliferation, migration, invasion, stemness, and chemoresistance of OSCC cells. Taken together, the data presented herein suggest that EGFR inhibitors might inhibit the malignant potential of OSCC cells through direct inhibition of not only EGFR downstream signaling pathway but also cellular uptake of OSCC cell-derived exosomes through macropinocytosis.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Exosomes; Humans; Mouth Neoplasms; Neoplastic Stem Cells; Pinocytosis; Protein Kinase Inhibitors; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Topics: B7-H1 Antigen; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Interferon-gamma; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Programmed Cell Death 1 Receptor; Squamous Cell Carcinoma of Head and Neck

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Lewis y is expressed in oral squamous cell carcinoma (OSCC) cells and tumors. Previously, we reported that Lewis y was not expressed in invasion areas, and attenuation of proliferation and invasion in OSCC cells was caused by over-expression of Lewis y. However, the roles of Lewis y in the attenuation of malignant properties have not been clarified. In this study, we investigated the roles of Lewis y in OSCC.. The levels of Lewis y on EGFR and the phosphorylation levels of EGFR in OSCC cells were analyzed by immunoprecipitation and western blot. EGFR cross-linking and binding kinetics of EGF were performed.. Upon EGF stimulation, phosphorylation and dimer formation of EGFR were more prominent in Lewis y- cells. EGF binding kinetics showed reduced binding sites in Lewis y+ cells.. Lewis y reduced EGF binding to EGFR, leading to suppression of malignant properties through suppression of EGF signaling.

Topics: Binding Sites; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lewis Blood Group Antigens; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Protein Binding; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancer in the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared with that of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.

Topics: Calcium, Dietary; Carcinoma, Squamous Cell; Catenins; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Head and Neck Neoplasms; Mouth Neoplasms; Phospholipase C gamma; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Epithelial-to-mesenchymal transition (EMT), a key step in oral cancer progression, is associated with invasion, metastasis, and therapy resistance, thus targeting the EMT represents a critical therapeutic strategy for the treatment of oral cancer metastasis. Our previous study showed that Abrus agglutinin (AGG), a plant lectin, induces both intrinsic and extrinsic apoptosis to activate the tumor inhibitory mechanism.. This study aimed to investigate the role of AGG in modulating invasiveness and stemness through EMT inhibition for the development of antineoplastic agents against oral cancer.. The EMT- and stemness-related proteins were studied in oral cancer cells using Western blot analysis and fluorescence microscopy. The potential mechanisms of Snail downregulation through p73 activation in FaDu cells were evaluated using Western blot analysis, immunoprecipitation, confocal microscopy, and molecular docking analysis. Immunohistochemical staining of the tumor samples of AGG-treated FaDu-xenografted nude mice was performed.. At the molecular level, AGG-induced p73 suppressed Snail expression, leading to EMT inhibition in FaDu cells. Notably, AGG promoted the translocation of Snail from the nucleus to the cytoplasm in FaDu cells and triggered its degradation through ubiquitination. In this setting, AGG inhibited the interaction between Snail and p73 in FaDu cells, resulting in p73 activation and EMT inhibition. Moreover, in epidermal growth factor (EGF)-stimulated FaDu cells, AGG abolished the upregulation of extracellular signal-regulated kinase (ERK)1/2 that plays a pivotal role in the upregulation of Snail to regulate the EMT phenotypes. In immunohistochemistry analysis, FaDu xenografts from AGG-treated mice showed decreased expression of Snail, SOX2, and vimentin and increased expression of p73 and E-cadherin compared with the control group, confirming EMT inhibition as part of its anticancer efficacy against oral cancer.. In summary, AGG stimulates p73 in restricting EGF-induced EMT, invasiveness, and stemness by inhibiting the ERK/Snail pathway to facilitate the development of alternative therapeutics for oral cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Mice, Nude; Molecular Docking Simulation; Mouth Neoplasms; Neoplastic Stem Cells; Plant Lectins; Snail Family Transcription Factors; Tumor Protein p73; Ubiquitination; Up-Regulation; Xenograft Model Antitumor Assays

The protein voltage-gated sodium channel Nav1.5 is highly upregulated in various types of cancer and, in general, promotes cancer cell invasiveness and metastatic progression. A previous study found that Nav1.5 was highly expressed in poorly differentiated oral squamous cell carcinoma (OSCC). However, whether Nav1.5 enhances invasiveness and metastasis of OSCC are still unknown. In this study, we found that Nav1.5 was highly expressed in OSCC cell lines compared with normal oral keratinocyte HOK cell line by using western blot analysis. CCK-8 assay results revealed that downregulation of Nav1.5 expression by its specific siRNA reduced proliferation of OSCC HSC-3 cells. Moreover, transwell assay results showed Nav1.5 knockdown significantly inhibited migration and invasion of HSC-3 cells. Meanwhile, qRT-PCR and western blot analysis results showed that epidermal growth factor (EGF) induced Nav1.5 expression in a time- and dose-dependent manner. In addition, EGF promoted proliferation, migration and invasion of HSC-3 cells. Importantly, the Nav1.5 inhibitor tetrodotoxin significantly inhibited the proliferation of HSC-3 cells and impeded the migration and invasion of HSC-3 cells. Furthermore, it was found that siRNA-mediated knockdown of Nav1.5 also lessened the proliferation of HSC-3 cells and blocked the migration and invasion of HSC-3 cells. Taken together, these results indicate that Nav1.5 is involved in the progression of OSCC and Nav1.5 promotes the proliferation, migration and invasion of OSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; RNA Interference; Sodium Channel Blockers; Tetrodotoxin

Epidermal growth factor (EGF) promotes tumourigenesis and tissue repair of epithelial and mesenchymal cells and has a role in chemotaxis, mitogenesis, cell motility, and cytoprotection. It also enhances the growth of cancers. EGF may therefore have a role in the initiation or promotion of oral carcinogenesis. The cases of 152 patients with oral squamous cell carcinoma whose preoperative serum EGF level was determined by enzyme-linked immunosorbent assay were analyzed retrospectively, along with those of 40 age- and sex-matched controls. Patients with higher levels of EGF were more likely to have neck lymph node metastasis (P=0.026), advanced stage cancer (P=0.04), and a worse survival status (P=0.0019). Multivariate analysis using the Cox proportional hazards model indicated that the EGF level was an independent predictor of poor survival (hazard ratio 1.99, P=0.018). Patients with higher preoperative serum EGF levels had significantly poorer cancer-specific survival by Kaplan-Meier analysis (P=0.032). This study indicates that a higher preoperative serum EGF level is associated with neck lymph node metastasis, more advanced stage, and poor survival. EGF should be considered as a potential prognostic biomarker and a therapeutic target for patients with oral cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Prognosis; Retrospective Studies; Survival Rate

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

Topics: Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Extracellular Vesicles; Humans; Mouth Neoplasms

Activated platelets release functional, high m.w. epidermal growth factor (HMW-EGF). In this study, we show platelets also express epidermal growth factor (EGF) receptor (EGFR) protein, but not ErbB2 or ErbB4 coreceptors, and so might respond to HMW-EGF. We found HMW-EGF stimulated platelet EGFR autophosphorylation, PI3 kinase-dependent AKT phosphorylation, and a Ca

Topics: Autocrine Communication; B-Cell Lymphoma 3 Protein; Blood Platelets; Carcinogenesis; Carcinoma, Squamous Cell; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Interleukin-1beta; Mouth Neoplasms; Neoplasm Invasiveness; Platelet Activation; Proto-Oncogene Proteins; Transcription Factors

Overexpression and increased signaling from the epidermal growth factor receptor (EGFR) often changes oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed the roles of OSCC-derived extracellular vesicles (EVs), including exosomes in the trafficking of cetuximab and in epithelial-mesenchymal transition (EMT) of epithelial cells. OSCC cells abundantly expressed EGFR, which was secreted from cells with OSCC-EVs upon EGF stimulations. The OSCC-EGFR-EVs were then able to enter into and transform epithelial cells leading to increased mesenchymal traits with increased vimentin and spindle-like shapes. EGF priming of OSCC cells further increased this EMT-initiating effect of the OSCC-EVs. The internalization and pro-EMT effects of the OSCC-EVs were largely blocked by cetuximab. Thus, OSCC-derived EVs transform normal epithelial cells into a mesenchymal phenotype and anti-EGFR therapeutic antibody cetuximab inhibits such a carcinogenic effect of the OSCC-EVs.

Topics: Cell Line, Tumor; Cell Transformation, Neoplastic; Cetuximab; Epidermal Growth Factor; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Exosomes; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Inhibiting BRD4 has emerged as a promising anticancer strategy, and inhibitors such as JQ1 can suppress cell growth in oral squamous cell carcinoma (OSCC). However, the mechanism through which JQ1 exerts its anticancer activity has not been reported. Moreover, JQ1 does not markedly inhibit proliferation and increase apoptosis in OSCC when used as a monotherapy. Herein, we explore the mechanism of JQ1 in OSCC and probe ways to increase its therapeutic potential. In this study, we used two cell lines, Cal27, and Scc25. We found that BRD4 was highly expressed in OSCC tissues when compared with adjacent non-tumor tissues, and JQ1 worked through the EGFR-mediated signaling pathway in tumor cells. Furthermore, we demonstrated that JQ1 induced an increased treatment effect in vitro and in vivo when combined with a PI3K inhibitor. Interestingly, subsequent mechanistic analyses indicated that further suppressing EGFR and BRD4 expression was instrumental to this functional synergism. Moreover, we found that upregulating EGFR expression by EGF stimulation protected cells treated with JQ1 from apoptosis, while knockdown of EGFR before addition of JQ1 successfully mimicked the combination treatment results. In summary, our findings revealed that JQ1 can act by inhibiting the EGFR-mediated signaling pathway, and EGFR expression influences the sensitivity of OSCC to JQ1. Regarding clinical use, this study demonstrates that BRD4 is a novel therapeutic target and EGFR can be used as a biomarker to identify the most appropriate anti-BRD4 treatment strategy in OSCC.

Topics: Aminopyridines; Analysis of Variance; Animals; Antineoplastic Agents; Apoptosis; Azepines; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Female; Gene Knockdown Techniques; Humans; Mice; Morpholines; Mouth Neoplasms; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Transcription Factors; Triazoles; Xenograft Model Antitumor Assays

The activation of receptor tyrosine kinases (RTKs) results in cellular effects including cell proliferation, survival, migration and invasion; RTKs also play an important role in tumourigenesis. It has been reported that EGFR signalling controls the migration of oral squamous cell carcinoma (OSCC) SAS and HSC3 cells but not of HSC4 cells, although the proliferation of HSC4 cells is regulated by EGF/EGFR. In the present study, we investigated the roles of EGFR and the c-Met signalling pathway in cell migration via filopodia and lamellipodia formation, which may be prerequisites for migration. To explore the role of c-Met in cell migration, we inhibited c-Met RTK activity using the c-Met inhibitor SU11274 and activated c-Met using hepatocyte growth factor (HGF) in three OSCC cell lines HSC4, SAS and Ca9-22 and investigated migration potency using a wound healing assay. We showed that inhibition of c-Met significantly suppressed, and activation of c-Met significantly promoted, the migration of OSCC cells. Additionally, the migration of SAS and Ca9-22 cells was inhibited by the EGFR inhibitors AG1478 and cetuximab and promoted by EGF treatment. Moreover, migration potency was correlated with lamellipodia formation. Furthermore, western blot analyses demonstrated that SU11274 decreased and HGF increased lamellipodin protein levels as well as phosphorylated c-Met levels. Collectively, we demonstrated that c-Met signalling induced lamellipodia formation by upregulating lamellipodin, thereby promoting the migration of OSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Indoles; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pseudopodia; Quinazolines; Signal Transduction; Sulfonamides; Tyrphostins

Topics: Acneiform Eruptions; Animals; Antineoplastic Agents, Immunological; Antiparasitic Agents; Biopsy; Cetuximab; Diagnosis, Differential; Epidermal Growth Factor; Humans; Ivermectin; Male; Middle Aged; Mites; Mouth Neoplasms

"Warburg effect", the enhanced glycolysis or aerobic glycolysis, confers cancer cells the ability to survive and proliferate even under stressed conditions. In this study, we explored the role of epidermal growth factor (EGF) in orchestrating Warburg effect, the epithelial-mesenchymal transition (EMT) process, and the acquisition of cancer stem-like cell properties in human oral squamous cell carcinoma (OSCC) cells. Our results showed that EGF induces EMT process in OSCC cells, which correlates with the acquisition of cancer stem-like properties, including the enrichment of CD44+/CD24- population of cancer cells and an increased expression of CSC-related genes, aldehyde dehydrogenase-1 (ALDH1) and Bmi-1. We also showed that EGF concomitantly enhanced L-lactate production, while blocking glycolysis by 2-deoxy-D-glucose (2-DG) robustly reversed EGF-induced EMT process and CSC-like properties in OSCC cells. Mechanistically, we demonstrated that EGF promoted EMT process and CSC generation through EGFR/PI3K/HIF-1α axis-orchestrated glycolysis. Using an orthotopic tumor model of human OSCC (UM-SCC1) injected in the tongue of BALB/c nude mice, we showed that treatment with 2-DG in vivo significantly inhibited the metastasis of tumor cells to the regional cervical lymph nodes and reduced the expression of ALDH1 and vimentin in both in situ tumors and tumor cell-invaded regional lymph nodes. Taken together, these findings have unveiled a new mechanism that EGF drives OSCC metastasis through induction of EMT process and CSC generation, which is driven by an enhanced glycolytic metabolic program in OSCC cells.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; CD24 Antigen; Cell Line, Tumor; Deoxyglucose; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Glycolysis; Head and Neck Neoplasms; Humans; Hyaluronan Receptors; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; Lactic Acid; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplastic Stem Cells; Phenotype; Phosphatidylinositol 3-Kinase; Polycomb Repressive Complex 1; Retinal Dehydrogenase; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Time Factors; Xenograft Model Antitumor Assays

The initiation and progression of cancer is closely associated with the tumor microenvironment. The overexpression of oncogenes during tumor growth and progression by stromal stimuli can affect the aggressiveness of the cancer. In this study, in vitro and in vivo studies were performed to examine the role of stromal epidermal growth factor (EGF) in enhancing the invasive potential of in low-invasive cancer. EGF was tested in order to elucidate the specific molecules that participate in increasing the invasive potential of low-invasive cancer cells. EGF stimulation enhanced cancer invasion in an EGF receptor (EGFR)-dependent manner. EGF induced insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) and podoplanin (PDPN) expression, which play an important role in oral squamous cell carcinoma (OSCC) cell invasion. An apparent tumor mass was not observed in the mouse xenograft; however, multiple tumor microfoci were seen in mice injected with IMP-3-overexpressing cells. These results show that EGF stimulates IMP-3 expression, thereby increasing cancer invasion and tumor progression.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Mice; Mice, Inbred BALB C; Mouth Neoplasms; Neoplasm Invasiveness; RNA-Binding Proteins; RNA, Messenger; Tumor Microenvironment

CD166 has been considered a relatively specific marker of stem cells and cancer stem cells, and the altered expression of CD166 has also been reported as a prognostic marker of several other types of cancer. However, the molecular functions of CD166 in these cancer cells are largely unknown. In this study, we found that CD166 significantly enhanced epidermal growth factor receptor (EGFR) phosphorylation and prolonged epidermal growth factor (EGF)/EGFR signalling activation. In addition, EGF stimulation in CD166-overexpressing oral squamous carcinoma cells led to enhanced colony formation, invasion capacity and cytoskeletal re-organization in vitro and elevated tumourigenesis in vivo. Taken together, the results of our study identify CD166 as an intriguing therapeutic target for patients suffering from oral squamous cell carcinoma (OSCC).

Topics: Antigens, CD; Carcinoma, Squamous Cell; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Fetal Proteins; Humans; Mouth Neoplasms; Phosphorylation; Signal Transduction

Oral lichen planus (OLP) is classified as a potential malignant disorder, and epidermal growth factor (EGF) may play a key role in cancer development. The aim of this study was to compare serum and saliva EGF among patients with OLP and oral squamous cell carcinoma (OSCC). A cross-sectional study was performed on 27 patients with OLP (10 reticular and 17 atrophic-erosive forms), 27 patients with OSCC and 27 healthy control group. The study was conducted at the Cancer Department, Clinic of Oral Medicine, Tehran University of Medical Sciences. The serum and saliva EGF were assayed by ELISA method. Statistical analysis of ANOVA was used. The mean serum EGF in OLP and OSCC patients was significantly lower compared to healthy control group (P<0.05), but no significant difference was observed between OLP and OSCC patients. There was no significant difference in mean salivary EGF among groups. As serum EGF levels appear to be statistically similar in OLP and OSCC, it seems that EGF might play a role in the pathogenesis of OLP and its cancerization.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Cross-Sectional Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Iran; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Saliva

Oral squamous cell carcinoma (OSCC) is one of the most prevalent carcinomas worldwide. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and modulate physiological or pathological processes including OSCC carcinogenesis. miR-31 has been found to be up-regulated in OSCC and to act as an oncogenic miRNA. However, the molecular mechanism underlying miR-31 up-regulation in OSCC is still obscure. The activation of epidermal growth factor receptor (EGFR) signaling axis plays key roles in driving oral carcinogenesis. Our screening identified that there is up-regulation of miR-31, miR-181b and miR-222 in OSCC cells following EGF treatment. Subsequent analysis showed that EGF treatment led to AKT activation, which then resulted in miR-31 up-regulation. Moreover, EGF treatment and the AKT activation induced by exogenous expression up-regulated C/EBPβ expression. The miR-31 up-regulation induced by EGF was abrogated by AKT inhibition or by the knockdown of C/EBPβ expression. In OSCC cell subclones stably overexpressing the functional isoform of C/EBPβ, miR-31 expression was up-regulated. Curcumin is a natural ingredient exhibiting anti-cancer potential. It was found that curcumin attenuated AKT activation and the up-regulation of C/EBPβ and miR-31 caused by EGF stimulation in OSCC cells. Lastly, concordance across the expression of EGFR, the expression of C/EBPβ and the expression of miR-31 in OSCC tissues was found. This study describes a novel scenario where the up-regulation of miR-31 expression in OSCC is, at least in part, a consequence of EGFR oncogenic activation. Although the AKT activation and C/EBPβ expression after EGF treatment might not be directly linked, both events are the crucial mediators underlying miR-31 up-regulation in the EGFR signaling axis.

Topics: Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Curcumin; Epidermal Growth Factor; Humans; MicroRNAs; Mouth Mucosa; Mouth Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

Oral and oropharyngeal cancer together constitute the sixth most common cancer worldwide, with over 400,000 new cases diagnosed each year. Early detection is paramount, as the 5-year survival rate for these cancers decreases markedly once tumors have become regionally invasive. In many tissues, including oral epithelia, neoplastic progression is accompanied by alterations in expression of the epithelial cell adhesion molecules E-cadherin and P-cadherin. Oral epithelia is one of only a few tissues in which P-cadherin levels have been noted to increase in dysplasia and well-differentiated carcinomas and decrease in advanced malignancies. In the present study, P-cadherin was overexpressed in both dysplastic and malignant oral keratinocytes to characterize the mechanisms by which aberrantly expressed P-cadherin may modulate tumor progression. We found that P-cadherin was able to potentiate ligand-dependent signaling of insulin-like growth factor 1 receptor (IGF-1R) in malignant keratinocytes and epidermal growth factor receptor (EGFR) in dysplastic cells. P-cadherin prolonged activation of the mitogen-activated protein kinase (MAPK) in both cell lines and also increased the magnitude of AKT phosphorylation in dysplastic cells. P-cadherin overexpression alone was sufficient to increase steady-state levels of the mesenchymal transcription factor Snail, increase cell motility and also induce morphological changes in dysplastic keratinocytes. Taken together, these data suggest that the aberrantly elevated levels of P-cadherin which occur in early oral tumor development may play a critical role in the augmentation of neoplastic signaling networks and in the further acquisition of aggressive phenotypes.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Cell Shape; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Keratinocytes; MAP Kinase Signaling System; Mouth Mucosa; Mouth Neoplasms; Receptor, IGF Type 1; Snail Family Transcription Factors; Transcription Factors

Phenethyl isothiocyanate (PEITC) is a natural compound that is involved in chemoprevention as well as inhibition of cell growth and induction of apoptosis in several types of cancer cells. Previous studies have revealed that PEITC suppresses the invasion of AGS gastric and HT-29 colorectal cancer cells. However, the effects of PEITC on the metastasis of SAS oral cancer cells remain to be determined. Our results showed that PEITC treatment inhibited the invasion of EGF-stimulated SAS cells in a concentration-dependent manner, but appeared not to affect the cell viability. The expression and enzymatic activities of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) were suppressed by PEITC. Concomitantly, we observed an increase in the protein expression of both tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2 (TIMP-2) in treated cells. Furthermore, PEITC treatments decreased the protein phosphorylation of epidermal growth factor receptor (EGFR) and downstream signaling proteins including PDK1, PI3K (p85), AKT, phosphorylated IKK and IκB to inactivate NF-κB for the suppression of MMP-2 and MMP-9 expression. In addition, PEITC can trigger the MAPK signaling pathway through the increase in phosphorylated p38, JNK and ERK in treated cells. Our data indicate that PEITC is able to inhibit the invasion of EGF-stimulated SAS oral cancer cells by targeting EGFR and its downstream signaling molecules and finally lead to the reduced expression and enzymatic activities of both MMP-2 and MMP-9. These results suggest that PEITC is promising for the therapy of oral cancer metastasis.

Topics: Anticarcinogenic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Humans; I-kappa B Kinase; Isothiocyanates; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mouth Neoplasms; Neoplasm Invasiveness; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

Expression of mucosa-associated lymphoid tissue 1 (MALT1) is inactivated in oral carcinoma patients with worse prognosis. However, the role in carcinoma progression is unknown. Unveiling genes under the control of MALT1 is necessary to understand the pathology of carcinomas.. Gene data set differentially transcribed in MALT1-stably expressing and -marginally expressing oral carcinoma cells was profiled by the microarray analysis and subjected to the pathway analysis. Migratory abilities of cells in response to MALT1 were determined by wound-healing assay and time-lapse analysis.. Totally, 2933 genes upregulated or downregulated in MALT1-expressing cells were identified. The subsequent pathway analysis implicated the inhibition of epidermal growth factor and transforming growth factor-β signalling gene expression, and highlighted the involvement in the cellular movement. Wound closure was suppressed by wild-type MALT1 (66.4%) and accelerated by dominant-negative MALT1 (218.6%), and the velocities of cell migration were increased 0.2-fold and 3.0-fold by wild-type and dominant-negative MALT1, respectively.. These observations demonstrate that MALT1 represses genes activating the aggressive phenotype of carcinoma cells, and suggest that MALT1 acts as a tumour suppressor and that the loss of expression stimulates oral carcinoma progression.

Topics: Caspases; Cell Line, Tumor; Cell Movement; Disease Progression; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lymphoid Tissue; Mouth Neoplasms; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Neoplasm Proteins; NF-kappa B; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24 h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50 % more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.

Topics: Alkaline Phosphatase; Animals; Apoptosis; Bacterial Proteins; Carcinoma; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; Extracellular Matrix Proteins; Gene Transfer Techniques; Genetic Vectors; Humans; Luminescent Proteins; Mice; Mice, Inbred BALB C; Mouth Neoplasms; Recombinant Proteins; Transfection

Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti-tumour signal inhibitors on the migratory activity.. Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time-lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting.. In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose-dependent activation of cell migration. Addition of the EGFR-specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF-induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI-3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF-induced migration was particularly sensitive to PI-3 K-inhibition, while in C12 cells, both HGF- and EGF-induced migration were highly sensitive to p38-blockade.. The results demonstrate that the MEK/ERK, p38 and PI-3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.

Topics: Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; MAP Kinase Signaling System; Mouth Neoplasms; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Signal Transduction; Tumor Cells, Cultured

Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21(cip1/waf1). In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.

Topics: 4-Nitroquinoline-1-oxide; Administration, Oral; Administration, Topical; Animals; Antineoplastic Agents; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chemoprevention; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Humans; Male; Molecular Targeted Therapy; Mouth Mucosa; Mouth Neoplasms; Polyethylene Glycols; Rats; Rats, Inbred F344

The effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.. A431 OSCC in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As(2)O(3) for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As(2)O(3) treatment. Next, mice with pharynx-implanted A431 cells received As(2)O(3) i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.. In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As(2)O(3) dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As(2)O(3) treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As(2)O(3) treatment in a dose responsive pattern.. This study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As(2)O(3) treatment.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Biomarkers, Tumor; Body Weight; Carbocyanines; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Injections, Intraperitoneal; Injections, Intravenous; Magnetic Resonance Imaging; Mice; Mice, Nude; Mouth Neoplasms; Neoplasms, Experimental; Oxides; Recombinant Fusion Proteins; Spectroscopy, Near-Infrared; Tumor Burden; Tumor Cells, Cultured

Human podoplanin is a type-1 transmembrane sialomucin-like glycoprotein that is involved in cell migration, tumor cell invasion and metastasis. Our recent study of oral squamous cell carcinoma (OSCC) demonstrated that the degree of immunohistochemical expression of podoplanin was correlated with the severity of epithelial dysplasia and significantly associated with a poor pathologic grade of differentiation. Furthermore, it has been reported that Src directly associates with the epidermal growth factor receptor (EGFR) in OSCC cells upon stimulation with EGF and phosphorylates Crk-associated substrate (Cas), podoplanin acting downstream of Src and Cas to promote cell migration. However, the molecular function of podoplanin remains unclear. In this study we performed real-time RT-PCR, Western blotting and scratch assay using OSCC cell lines in order to clarify the molecular biological function of podoplanin expression associated with various growth factors including EGF and with the Src-Cas signaling pathway. Podoplanin was found to have a marked influence on cancer cell migration and the expression of matrix metalloprotease-9 (MMP-9) in the oral cavity upon stimulation with EGF. Podoplanin promotes oral cancer cell migration, and the EGF-Src-Cas pathway is one of the possible mechanisms responsible for progression of cancer in the oral cavity.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Crk-Associated Substrate Protein; Epidermal Growth Factor; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Membrane Glycoproteins; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Microenvironment

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

Topics: Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Extracellular Matrix Proteins; Humans; Kalinin; Laminin; Mouth Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta1; Vimentin

The objective of this research was to investigate the salivary levels of epidermal growth factor (EGF) in patients with oral squamous cell carcinoma (OSCC) in comparison with clinically healthy individuals and to verify the immunoexpression of EGF in tumor samples. In addition, the relationship between salivary levels and tumoral EGF expression with clinicopathologic features was investigated. We carried out an investigation on EGF expression in lesion samples and in saliva of OSCC patients through immunohistochemistry and enzyme-linked immunosorbent assay, respectively. EGF salivary levels of OSCC patients were also compared with levels in saliva of healthy controls. EGF levels were significantly lower in OSCC patients in comparison with the control group. Smoking, tumor location, and alcohol consumption affected salivary levels of EGF. Strong immunoexpression of EGF was associated with a more aggressive histologic pattern of the lesion. There was no significant association among salivary levels and immunohistochemical expression of EGF. Although EGF expression is frequently observed in tumors, salivary levels of EGF are reduced in patients with OSCC samples. Tobacco and alcohol may decrease EGF in saliva, which may contribute to oral carcinogenesis. Indeed, further investigations are needed to elucidate the EGF pathways.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alcohol Drinking; Biomarkers; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Mouth; Mouth Neoplasms; Prognosis; Risk Factors; Saliva; Smoking

The erbB receptors and their ligands are involved in the pathogenesis and progression of oral squamous cell carcinoma (OSCC). Although EGFR and Her-2 are frequently overexpressed in OSCC, few studies evaluated these proteins in saliva and their association with the tumor, which may represent potential usefulness in a clinical setting.. The levels of EGFR, Her-2, and EGF were evaluated in saliva of 46 patients with OSCC before and after the surgical removal of the lesion, as well as in matched healthy controls. The relationship of salivary levels and EGFR and Her-2 immunoexpression in tumor samples with clinicopathological features was analyzed.. EGFR and Her-2 salivary levels did not show difference between to pre-surgery and control groups, however, both demonstrated an increase after surgical removal of the tumor. No association was detectable among receptor salivary levels, tissue expression and clinicopathological features. EGF levels in pre-surgery group were significantly lower when compared to the control group.. EGFR and Her-2 were not considered to be valuable salivary tumor markers in OSCC, however, lower levels of EGF in saliva may suggest a higher susceptibility for OSCC development.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Mouth Neoplasms; Receptor, ErbB-2; Saliva

We designed an epidermal growth factor (EGF)-containing polyamidoamine (PAMAM) Generation 4 dendrimer vector labeled with quantum dots for targeted imaging and nucleic acid delivery. (1)H NMR, SDS-PAGE, and Western blotting were applied to characterize the synthesized G4.0-GGG-EGF nanoparticles. Targeting efficiency, cell viability, proliferation, and intracellular signal transduction were evaluated using HN12, NIH3T3, and NIH3T3/EGFR cells. We found that EGF-conjugated dendrimers did not stimulate growth of EGFR-expressing cells at the selected concentration. Consistent with this, minimal stimulation of post-receptor signaling pathways was observed. These nanoparticles can localize within cells that express the EGFR in a receptor-dependent manner, whereas uptake into cells lacking the receptor was low. A well characterized vimentin shRNA (shVIM) and yellow fluorescent protein (YFP) siRNA were used to test the delivery and transfection efficiency of the constructed targeted vector. Significant knockdown of expression was observed, indicating that this vector is useful for introduction of nucleic acids or drugs into cells by a receptor-targeted mechanism.

Topics: Animals; Dendrimers; Epidermal Growth Factor; ErbB Receptors; Humans; Magnetic Resonance Imaging; Mice; Mouth Neoplasms; Nanoparticles; NIH 3T3 Cells; Nucleic Acids; Signal Transduction

Topics: beta-Defensins; Biomarkers; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Erythroplasia; Humans; Leukoplakia, Oral; Mouth Neoplasms; Precancerous Conditions; Prognosis; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Human beta-defensins (hBDs) are small, cationic antimicrobial peptides produced by oral and other mucosal epithelia. More recently, hBDs have been shown to regulate adaptive immunity. In this study, we provide new information about the potential role of hBD-3 in the progression of oral cancer. In normal human oral epithelia, hBD-3 is produced by mitotically active cells in the basal layers of oral epithelium, whereas hBD-1 and -2 are coexpressed in the differentiated spinosum and granulosum layers. Interestingly, premalignant cells in carcinoma in situ lesions overexpress hBD-3, but not hBD-1 and hBD-2, correlating with specific recruitment and infiltration of macrophages. Our in vitro studies demonstrate that hBD-3 chemoattracts THP-1 monocytic cells and that epidermal growth factor (EGF) significantly induces hBD-3 expression in oral epithelial cells via mitogen-activated protein kinase (MAPK) kinase MEK1/2, p38 MAPK, protein kinase C (PKC), and phosphoinositide 3 kinase (PI3K), but not via Janus kinase (JAK) and signal transducer and activator of transcription (STATs). These results suggest that hBD-3 serves as a mitogen responsive gene in the initiation of oral cancer and may act as a motility signal to recruit tumor-associated macrophages.

Topics: beta-Defensins; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Humans; Macrophages; Microscopy, Fluorescence; Mouth Neoplasms; Phosphotransferases; Precancerous Conditions; STAT Transcription Factors

Snail is a regulator of epithelial-mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (alpha SMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGF beta 1 and EGF on Snail, E-cadherin, vimentin, and alpha SMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to alpha SMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by alpha SMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.

Topics: Actins; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Epidermal Growth Factor; Fibroblasts; Humans; Keratins; Middle Aged; Mouth Neoplasms; Myoblasts; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta1; Vimentin

nm23-H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23-H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23-H1. In our experiments, cells overexpressing nm23-H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23-H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23-H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23-H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Flow Cytometry; Humans; Immunohistochemistry; Immunoprecipitation; Integrin beta1; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Neoplasm Invasiveness; NM23 Nucleoside Diphosphate Kinases; Oligopeptides; Peptides; Protein Binding; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

It has been suggested that epithelial cyclooxygenase-2 (COX-2) promotes oral carcinogenesis and carcinoma malignancy through increased prostaglandin E(2) (PGE(2)) production. Although oral squamous cell carcinomas (OSCC) often express COX-2, they may also produce PGE(2) in a COX-1-dependent manner. We used 6 isolated cell lines to investigate which COX isoforms OSCC may use for PGE(2) production. COX-1 and -2 expression patterns divided the 6 OSCC cell lines into 3 distinct groups: both COX isoforms low, only COX-1 high, or both COX isoforms high. Multicolor immunohistofluorescence staining confirmed the COX-expression profiles in organotypic 3D cultures and the COX-2 dominance in OSCC tumors. Epidermal growth factor (EGF) stimulation induced COX-2 (but not COX-1) expression and increased PGE(2) production, which was attenuated by COX-2 (but not COX-1) specific inhibition or siRNA-mediated COX-2 gene knockdown. Thus, PGE(2) production in OSCC cell lines was COX-2-dependent.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Epidermal Growth Factor; Female; Gene Expression; Gene Knockdown Techniques; Humans; Intramolecular Oxidoreductases; Isoenzymes; Male; Middle Aged; Mouth Neoplasms; Prostaglandin-E Synthases; RNA, Small Interfering

The development of noninvasive molecular imaging approaches has the potential to improve management of cancer.. In this study, we demonstrate the potential of noninvasive topical delivery of an epidermal growth factor-Alexa 647 (EGF-Alexa 647) conjugate to image changes in epidermal growth factor receptor expression associated with oral neoplasia. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of EGF-Alexa 647 in a variety of model systems, including cells, three-dimensional tissue cultures, and intact human tissue specimens using wide-field and high-resolution fluorescence imaging. Data were collected from 17 different oral cancer patients: eight pairs of normal and abnormal biopsies and nine resected tumors were examined.. The EGF-dye conjugate can be uniformly delivered throughout the oral epithelium with a penetration depth exceeding 500 microm and incubation time of less than 30 minutes. After EGF-Alexa 647 incubation, the presence of oral neoplasia is associated with a 1.5- to 6.9-fold increase in fluorescence contrast compared with grossly normal mucosa from the same patient with both wide-field and high-resolution fluorescence imaging.. Results illustrate the potential of EGF-targeted fluorescent agents for in vivo molecular imaging, a technique that may aid in the diagnosis and characterization of oral neoplasia and allow real-time detection of tumor margins.

Topics: Animals; Cell Line, Tumor; Cyclic AMP; Diagnostic Imaging; Epidermal Growth Factor; ErbB Receptors; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mouth Neoplasms; Reproducibility of Results; Sensitivity and Specificity

The intracellular signalling cascade(s) mediating epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression is poorly defined in oral carcinomas. Investigation of two different oral squamous cell carcinoma (OSCC) cell lines with high EGF-induced COX-2 expression revealed, however, that this expression was dependent on two mitogen-activated protein kinase (MAPK) pathways [extracellular signal-regulated kinase 1/2 (ERK1/2) and p38] because combined inhibition of these pathways was needed to abolish EGF-induced COX-2 expression. Surprisingly, inhibition of phosphoinositide-3 kinase (PI3K) increased EGF-induced COX-2 expression in the basaloid OSCC cell line (C12), suggesting a PI3K-controlled, inhibitory COX-2-regulating pathway. Neither the transcription factor nuclear factor-kappaB (NF-kappaB), nor Src, was involved in EGF-induced COX-2 expression. The results suggest that EGF-induced COX-2 expression is regulated by several pathways, and emphasizes that individual tumors use different strategies for intracellular signalling.

Topics: Aged; Carcinoma, Basosquamous; Carcinoma, Squamous Cell; Carcinoma, Verrucous; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Enzymologic; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured

Epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression in squamous cell carcinomas is mediated through the extracellular signal-regulated kinase 1/2 and p38 pathways. Examination of a basaloid and a conventional oral squamous cell carcinoma cell line revealed that inhibition of c-Jun N-terminal kinase (JNK) with SP600125 increased EGF-induced (but not basal) COX-2 transcription 1.5-1.9-fold in extracellular signal-regulated kinase 1/2 and p38 pathway-dependent manners. Although JNK may phosphorylate the cyclosporine A-sensitive transcription factor, nuclear factor of activated T cells c3, it was seemingly not involved because cyclosporine A did not reduce EGF-induced COX-2 expression. Thus, JNK negatively regulated EGF-induced extracellular signal-regulated kinase 1/2 and/or p38-mediated COX-2 transcription, presumably through activating an unidentified phosphatase.

Topics: Anthracenes; Calcineurin Inhibitors; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 2; Cyclosporine; Dactinomycin; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; NFATC Transcription Factors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; Transcription, Genetic

Aberrant expression of N-cadherin is associated with tumor progression in squamous cell carcinomas (SCCs). Consequently, we examined the regulation of N-cadherin by TGFbeta1, an important mediator of keratinocyte and SCC function. N-cadherin expression was increased in oral SCC (OSCC) cell lines, regulating motility and correlating with TGFbeta1 production. Moreover, in normal keratinocytes TGFbeta1 increased expression of N-cadherin to regulate motility. TGFbeta1-mediated N-cadherin expression in the oral keratinocytes was blocked using siRNA targeting Smads. Unexpectedly, we found that EGF blocked TGFbeta1-mediated N-cadherin expression in oral keratinocytes and not in OSCC cells. Mechanistically, EGF enhanced Smad phosphorylation in the linker region, and attenuated TGFbeta1-mediated phosphorylation of Smad at the C-terminus, localization of Smad to the nucleus as well as Smad-driven promoter activity exclusively in oral keratinocytes but not in OSCC cells. The effect of EGF on TGFbeta1-mediated Smad-driven promoter activity and N-cadherin expression was reversed when activation of ERK1/2 was blocked. Although EGF and TGFbeta1 independently promoted migration of both oral keratinocytes and OSCC cells, EGF decreased TGFbeta1-mediated migration of oral keratinocytes but enhanced migration of OSCC cells. Together, these data support a model wherein EGF signaling has an important negative regulatory role on TGFbeta1-mediated N-cadherin expression and motility in normal oral keratinocytes, and in which loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Epithelium; Humans; Keratinocytes; Mitogen-Activated Protein Kinase 3; Mouth; Mouth Neoplasms; Signal Transduction; Transforming Growth Factor beta1; Tumor Cells, Cultured

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Drug Combinations; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotoxins; Models, Biological; Mouth Neoplasms; Saponins

Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Epidermal Growth Factor; Escherichia coli; Humans; Immunotoxins; Inhibitory Concentration 50; Mouth Neoplasms; Saponins; Time Factors

Epidermal growth factor (EGF) is excreted in a high concentration in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer growth and invasion, we analyzed the Matrigel invasion activity of the cultured oral cancer cell line. Cells grown under the influence of EGF were subjected to Matrigel invasion assays and cells grown in the absence of EGF were used as controls. Gelatin-zymography and Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay (CAT assay) determined the EGF stimulation of matrix metalloproteinase (MMP) expression. EGF increased the number of cells penetrating a Matrigel membrane. Gelatin-zymography and Northern blot analysis revealed that MMP9 and Ets1 expressions correlated with EGF but MMP2 was not changed. a transient transfection assay revealed that EGF increased the promoter activities of the MMP9 genes in HSC3 and SAS cells. These results suggest that EGF increases the invasion activity of oral cancer cells partly by increasing MMP9.

Topics: Biocompatible Materials; Blotting, Northern; Carcinoma, Squamous Cell; Chloramphenicol O-Acetyltransferase; Collagen; Drug Combinations; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Laminin; Matrix Metalloproteinase 9; Mouth Neoplasms; Neoplasm Invasiveness; Proteoglycans; Saliva; Tumor Cells, Cultured

We examined the expression of epiregulin and amphiregulin mRNA in 39 oral SCCs, 2 epithelial dysplasias and 7 normal gingivae by real-time RT-PCR. The mean expression level of epiregulin mRNA was higher in oral SCCs (0.29+/-0.50) than normal gingivae (0.01+/-0.007) and epithelial dysplasias (0.01+/-0.001). The expression level of epiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.023). Epiregulin mRNA was higher in stage III/IV than in stage I/II oral SCCs. However, a significant association was not found. The mean expression level of amphiregulin mRNA was higher in oral SCCs (0.18+/-0.24) than normal gingivae (0.002+/-0.003) and epithelial dysplasias (0.01+/-0.001). Amphiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.001). We then examined the expression of four EGF receptor mRNA in oral SCCs. The expression levels of HER1, HER2, HER3 and HER4 mRNA in oral oral SCCs were increased compared to those in normal gingivae. A significant correlation was found between the mRNA expression levels of epiregulin and HER2, HER3 and HER4 (Spearman's correlation coefficient by rank test, P=0.031, P=0.004 and P=0.027, respectively). Patients with oral SCC that have a high expression of epiregulin had a significantly shorter survival than those with low a expression (log-rank test, P<0.05). These results indicate that human epiregulin is closely linked to the increased or abnormal cell proliferation in human oral SCC.

Topics: Amphiregulin; Carcinoma, Squamous Cell; Cell Proliferation; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Ligands; Male; Mouth Neoplasms; Prognosis; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium.

Topics: Biopsy; Carcinoma; Cell Nucleus; Contrast Media; Cytoplasm; Epidermal Growth Factor; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mouth Neoplasms; Sensitivity and Specificity

Topics: Cancer Vaccines; Epidermal Growth Factor; Genetic Therapy; Humans; Mouth Neoplasms; Pharyngeal Neoplasms

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

Salivary epidermal growth factor (EGF) effect on oral cancer biology is unknown. We examined changes in minute volumes of whole resting and stimulated saliva, EGF concentration and its output (ELISA) in whole resting and stimulated saliva before and 2 weeks after surgical treatment in oral carcinoma patients compared to the control group. After stimulation salivary flow increased both in the control (P=0.003) and in the patients group--before (P=0.007) and after surgery (P=0.005). Higher stimulated saliva volume levels were observed before surgery than in post-treatment patients (P=0.032). A trend was seen with increasing EGF salivary concentrations after tumour excision both in resting (P=0.508) and stimulated (P=0.647) saliva. A similar ascending tendency of EGF output in stimulated saliva of post-treatment patients was observed (P=0.878). Decreased levels of EGF concentration in saliva before and its contrary tendency after surgical treatment may suggest an important role of EGF in oral cancer tumourogenesis.

Topics: Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Mouth Neoplasms; Postoperative Period; Saliva; Salivation

A suitable model analyzing the behavior of well-differentiated squamous cell carcinoma has not yet been established. We tried to establish such a system using a reconstructed oral mucosa, in which T3M-1 squamous cell carcinoma cells were cultured on 3T3 fibroblast-containing collagen gel. Fibroblasts promoted the stratification and keratinization of T3M-1 cells. During growth, the Ki-67 index of T3M-1 cells with fibroblasts was higher than that of T3M-1 cells alone. Fibroblasts increased the expression of involucrin, a differentiating marker of keratinocytes, in T3M-1 cells. They also promoted the invasion of T3M-1 cells into the gel. When T3M-1 cells alone were cultured in a fibroblast-conditioned (FC) medium, the fibroblast-induced phenomena mentioned above were almost replicated. In addition, epidermal growth factqr (EGF) promoted T3M-1 cells growth, but not the invasion. cDNA microarray analysis showed that FC medium increased the expression of EGF receptor and several other mRNAs of T3M-1 cells. The data suggest that T3M-1 cells, under cancer-stromal fibroblast interaction, undergo invasive growth with their well-differentiated squamous phenotype, and that this interaction may be mediated partly by soluble molecules (e.g., EGF) in an autocrine or paracrine pathway. Our system will probably provide a useful model for analyzing the biological behavior of well-differentiated squamous cell carcinoma.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Coculture Techniques; Collagen; Culture Media, Conditioned; Epidermal Growth Factor; Fibroblasts; Gels; Hepatocyte Growth Factor; Humans; Ki-67 Antigen; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Protein Precursors; Receptors, Interleukin-1; Stromal Cells

Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique, we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.

Topics: Antigen-Antibody Complex; Biomarkers, Tumor; Cell Line; Epidermal Growth Factor; Humans; Immunoassay; Keratinocytes; Molecular Probe Techniques; Mouth Neoplasms; Nanotubes; Staining and Labeling; Surface Plasmon Resonance

Oral epithelial tumour tissue, and cultured cervical epithelial carcinoma cells have been studied using synchrotron infrared microspectroscopy. Mid infrared absorption spectra collected at cellular spatial resolution from within oral tumours were found to be sufficiently distinct, when analysed by principal component analysis, to distinguish between three different cell types within the tumour. The resulting data were sufficiently robust to allow correct classification of spectra from cells within subsequent tissue samples. These results go some way to demonstrate the potential of infrared spectroscopy as a tool in the post-operative screening of oral cancer patients by the examination of exfoliated epithelial cells. To gain a better understanding of the inherent variability in the infrared spectra of such epithelial cells, we have studied A431 carcinoma cells under the stimulus of the growth-stimulating hormone EGF. We have detected key changes in the infrared spectrum that relate to the activation of the growth factor signalling mechanism.

Topics: Carcinoma; Cells, Cultured; Culture Techniques; Epidermal Growth Factor; Humans; Infrared Rays; Mouth Mucosa; Mouth Neoplasms; Spectrophotometry, Infrared; Synchrotrons

Discovery of the common and ubiquitous molecular targets for the disruption of angiogenesis, that are independent of the characteristics of malignant tumors, is desired to develop the more effective antitumor drugs. In this study, we propose that the platelet-derived growth factor receptor-alpha (PDGFRalpha)-p70S6K signal transduction pathway in mesenchymal cells, which is required for functional angiogenesis induced by fibroblast growth factor-2, is the potent candidate. Using murine limb ischemia as a tumor-free assay system, we demonstrated that p70S6K inhibitor rapamycin (RAPA) targets mesenchymal cells to shut down the sustained expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), via silencing of the PDGFRalpha-p70S6K pathway. Irrespective of the varied expression profiles of angiogenic factors in each tumor tested, RAPA constantly led the tumors to dormancy and severe ischemia in the time course, even associated with upregulated expression of VEGF from tumors. Because RAPA showed only a minimal effect to hypoxia-related expression of VEGF in culture, these results suggest that RAPA targets the host-vasculature rather than tumor itself in vivo. Thus, our current study indicates that the PDGFRalpha-p70S6K pathway is an essential regulator for FGF-2-mediated therapeutic neovascularization, as well as for the host-derived vasculature but not tumors during tumor angiogenesis, via controlling continuity of expression of multiple angiogenic growth factors.

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Hepatocyte Growth Factor; Hindlimb; Humans; Ischemia; Liver Neoplasms, Experimental; Male; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Nude; Mouth Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor alpha; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stromal Cells

Fatty acid synthase (FAS) is the enzyme responsible for the endogenous synthesis of saturated long-chain fatty acids from the precursors acetyl-CoA and malonyl-CoA. A growing body of evidence indicates that FAS is over expressed in several human cancers, such as prostate, breast, bladder, liver, lung, melanoma and oral squamous cell carcinoma (SCC). In the present study we used human oral SCC cell lines (SCC-4, -9, -15 and -25) as a model to investigate the role of FAS in the pathogenesis of oral cancer. RT-PCR and western blot experiments demonstrated that FAS is differentially expressed by the four oral SCC cell lines, with the highest production in SCC-9 followed by SCC-25. FAS expression in SCC-4 and -15 was similarly lower than the other cell lines. Proliferation curves and immunocytochemistry for PCNA and Ki-67 demonstrated that SCC-25 has the highest proliferative potential. In addition, the specific inhibitor of FAS activity cerulenin was able to significantly reduce the proliferation of oral SCC cells. Expression of androgen receptor was low in SCC-4, -9 and -15 and undetectable in SCC-25, whereas EGFR and c-erb-B2 were expressed in high amounts by the four cell lines. Immunocytochemical reactions showed that SCC-25 expresses higher levels of EGF compared to the other three cell lines. Finally, oral SCC cells exposed to nanomolar concentrations of exogenous EGF presented a reduction in the FAS protein levels concomitant with a decrease in their proliferation rates. Taken together, our results indicate that FAS is expressed in an apparently androgen-independent fashion in oral SCC cells and it is necessary for their proliferation.

Topics: Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Fatty Acid Synthases; Humans; Mouth Neoplasms; Receptor, ErbB-2; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

IGF-I receptor (IGF-IR) is involved in numerous biological functions via its major downstream signaling molecules, extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase/Akt. The IGF-I-induced activation of ERK, but not that of Akt, is reportedly mediated by the transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase (TK). The mechanism for the EGFR-TK-dependent activation, however, still remains largely unknown. We found that an oral carcinoma cell line overexpressing EGFR, Ca9-22, exhibited IGF-I-induced activation of both Akt and ERK, but that only the latter was significantly decreased by a specific inhibitor of EGFR-TK, tyrphostin AG1478. In this report we provide evidence for the existence in this cell line of a novel mechanism by which IGF-I induces ERK activation in a manner that is dependent on the basal level of EGFR-TK activity, but is independent of receptor transactivation. In addition, we show that c-Raf kinase is likely to be a key regulator of this mechanism. The elucidation of such a unique mechanism involving cross-talk between EGFR and heterologous receptors may shed additional light on the clinical use of EGFR-TK inhibitors in antitumor therapies.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; GRB2 Adaptor Protein; Heparin-binding EGF-like Growth Factor; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Mouth Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Quinazolines; ras Proteins; Receptor Cross-Talk; Receptor, IGF Type 1; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tyrphostins

Hepatocyte growth factor (HGF), the ligand for the c-met proto-oncogene product, is a multifunctional protein that enhances tumor cell motility, extracellular matrix invasion, and mitogenic or morphogenic activities of various cell types. In this study we examined the expression of the c-Met receptor in human oral squamous cell carcinoma (SCC) in vivo and in vitro to explore its relationship to tumor progression and invasiveness. Biopsy specimens of human oral SCC were immunohistochemically stained for c-Met. Nearly all primary oral SCC lesions and lymph node metastases consistently showed intense staining for c-Met, whereas normal oral mucosa showed faint to negative staining only on basal cells. In a panel of human oral SCC cell lines, we found a strong correlation between the levels of c-Met expression and the cells' response to HGF in motility and invasion assays. Sensitivity to HGF also correlated with the expression of the c-Met 9-kb mRNA. When the non-invasive HOC-605 cell line, which expresses a low level of c-Met receptor, was transfected with an expression plasmid containing human c-met cDNA, the transfectant cells showed motile and invasive responses to HGF. Immunostaining and immunoprecipitation studies demonstrated that E-cadherin and c-Met were physically associated at SCC cell-cell junctions, suggesting a direct role for c-Met in induction of junctional integrity. Importantly, HGF caused a rapid elevation of unbound beta-catenin, suggesting its availability for nuclear signal transduction and triggering of cell motility and invasiveness. Thus, overexpression of c-Met may facilitate disruption of E-cadherin junctions. Collectively, these results suggest that HGF/c-Met signaling is a common event in oral SCC that may trigger phenotype modulation and enhanced invasion and metastasis.

Topics: beta Catenin; Blotting, Northern; Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Intercellular Junctions; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met; Signal Transduction; Trans-Activators; Tyrosine

Sebaceous adenoma (SA) is a rare solitary tumour with a predilection for the forehead and scalp. In the English literature, less than 10 cases of SA have been described in the oral cavity. The objective of this study was to examine the clinicopathologic features and evaluate the expression of epidermal growth factor and its receptor, estrogen receptor and androgen receptor in SA and in its differential diagnoses including sebaceous gland hyperplasia (SGH) and Fordyce's granules (FG). Additionally, we analysed the proliferative potential of sebaceous cells from SA, SGH and FG by measuring proliferating cell nuclear antigen (PCNA) expression and quantification of argyrophilic nuclear organizer regions (AgNORs). The SA showed many clinicopathologic similarities to cases previously reported including the biphasic population of cells, in the periphery of lobules undifferentiated basaloid cells whereas the central area was formed by mature sebocytes. SA was composed of 198 lobules of sebaceous cells, whereas SGH and FG showed a mean of 21 +/- 7.81 and 5.84 +/- 2.83, respectively. The AgNOR and PCNA indices were similar in SA, SGH and FG. These data suggest that lobule counts may be used as additional criteria in distinguishing SA of the oral cavity from other intraoral sebaceous gland lesions.

Topics: Adenoma; Adolescent; Adult; Aged; Aged, 80 and over; Choristoma; Diagnosis, Differential; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hyperplasia; Middle Aged; Mouth Diseases; Mouth Neoplasms; Nucleolus Organizer Region; Proliferating Cell Nuclear Antigen; Receptors, Androgen; Receptors, Estrogen; Sebaceous Glands; Sweat Glands

Buccal mucosa carcinoma-derived cell line, HO-1-N-1, epithelial-like cells, was obtained in order to investigate the characteristics of oral cancer cells and examine the [Ca2+]i responses to stimulants, such as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha ). Intracellular Ca2+ influx was observed by all stimulants that enhanced the [Ca2+]i response. However, intracellular Ca2+ release was not observed in response to growth factors. The [Ca2+]i response of BK (100 nM) was inhibited by 10 micro M of the BKB2 antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK, and HIST (1 mM) was completely inhibited by 100 nM of the H1 antagonist, (+)-chlorpheniramine, in the presence and absence of extracellular Ca2+ (1.5 mM).

Topics: Bradykinin; Calcium; Calcium Signaling; Carcinoma; Epidermal Growth Factor; Epithelial Cells; Histamine; Histamine H1 Antagonists; Humans; Mouth Mucosa; Mouth Neoplasms; Receptors, Cell Surface; Stimulation, Chemical; Thapsigargin; Transforming Growth Factor alpha; Tumor Cells, Cultured

A doxorubicin and [D-Lys(6)]Luteinizing Hormone-Releasing Hormone (LH-RH) conjugate, AN-152, was designed to target LH-RH receptor positive cells. AN-152 is more potent against a specific group of cancers than doxorubicin and has less peripheral toxicity. This therapy is potentially efficacious against many other types of malignancies. Here, AN-152 was tested on oral (KB) and laryngeal (HEp-2) carcinoma cells. LH-RH receptor presence was demonstrated by displacement binding assays. Cells were treated with 10 nM EGF or left untreated, then exposed to 0-10 microM AN-152. Cytotoxicity was assessed by MTT assay to determine ED(50) values. AN-152 association with the cells was monitored using two-photon laser scanning microscopy. AN-152 was more potent than doxorubicin in both KB and HEp-2 cell lines (P<0.05). Up-regulation of LH-RH receptors by epidermal growth factor (EGF) increased the entry and potency of AN-152 in KB cells, but not in HEp-2 cells. This novel approach may be effective against a variety of cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Division; Doxorubicin; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Laryngeal Neoplasms; Mouth Neoplasms; Tumor Cells, Cultured

Invasive squamous cell carcinoma (SCC) cells degrade extracellular matrix (ECM) via an extracellular protease cascade that includes urokinase-type plasminogen activator (uPA), plasmin, and the metalloprotease (MMP) family of collagenases. In this study, treatment of oral SCC cells with epidermal growth factor (EGF) stimulated the cells to invade Matrigel (constructive basement membrane (BM) protein). EGF-induced cell invasion was inhibited by antibodies to uPA and by synthetic uPA inhibitors. EGF also induced increased expression of uPA and uPA receptor (uPAR) proteins and mRNA, as well as transcription factor activator protein-1 (AP-1)-DNA binding. These EGF-induced changes were inhibited by treatment with dexamethasone (DEX). DEX treatment also stimulated the production of plasminogen activator inhibitor type 1. Moreover, transfection of SCC cells with AP-1 decoy oligodeoxynucleotides (ODNs) resulted in the suppression of EGF-induced uPA and uPAR expression and Matrigel invasion. These results suggest that oral SCC cells invade Matrigel mainly through the uPA-plasmin cascade, which is mediated by the transcription factor AP-1. The uPA-uPAR interaction is essential for augmenting proteolytic activity and uPAR-mediated signaling, which ultimately induce motility and invasion. Since DEX inhibits the expression of both uPA and uPAR, it may be a useful treatment for oral SCC.

Topics: Carcinoma, Squamous Cell; Cell Movement; Dexamethasone; DNA; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Invasiveness; Oligodeoxyribonucleotides; Plasminogen Activator Inhibitor 1; RNA, Messenger; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

An oral epithelial cell line derived from buccal mucosa squamous cell carcinoma, HO-1-N-1, was used to elucidate the role of epidermal growth factor (EGF) in saliva on wound healing of the oral mucosa. The effects of EGF on DNA synthesis, and cell migration was studied and the related signal transduction pathways examined. DNA synthesis by HO-1-N-1 cells was stimulated dose-dependently by 1-10 ng ml(-1) EGF, but significantly inhibited by addition of a PI3-K inhibitor (wortmannin), a p38 MAPK inhibitor (SB203580) or an MEKs inhibitor (PD98059). Cell migration was also accelerated by addition of 1-10 ng ml(-1) EGF; however, the migration rate was decreased to 30% by adding PD98059, to 40% by adding a tyrosine kinase inhibitor (herbimycin A), and to 60% by adding wortmannin or dexamethasone. These results indicate that the physiologic concentration of EGF in saliva may stimulate proliferation and migration of oral epithelial cells for wound healing, when the oral mucosa has been injured. Furthermore, this study revealed that EGF-stimulated signal transduction pathways for epithelial cell proliferation and cell migration are different.

Topics: Androstadienes; Carcinoma, Squamous Cell; Cell Division; Cell Movement; Dexamethasone; DNA; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Glucocorticoids; Humans; Immunity, Mucosal; MAP Kinase Signaling System; Mouth Mucosa; Mouth Neoplasms; Saliva; Salivary Proteins and Peptides; Tumor Cells, Cultured; Wortmannin; Wound Healing

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.

Topics: Biopsy; Blotting, Western; Carcinoma, Squamous Cell; Collagen; Epidermal Growth Factor; Humans; In Situ Hybridization; Matrix Metalloproteinase 1; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Biopsy; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Histocompatibility Antigens Class I; Humans; Isoenzymes; Mouth Neoplasms; Neoplasms; Placenta; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms

Cyclooxygenase-2 (Cox-2), the inducible form of cyclooxygenase, is up-regulated in tumors and transformed cells. Because this enzyme catalyzes the formation of prostaglandins from arachidonic acid, chemopreventive strategies that suppress its expression could be useful for preventing cancer. We investigated whether retinoids suppressed basal expression of Cox-2 or EGF-mediated induction of Cox-2 in human oral squamous carcinoma cells. Treatment with retinoids [all-trans-retinoic acid (all-trans-RA), 9-cis-RA, 13-cis-RA, and retinyl acetate] suppressed both basal levels of Cox-2 and EGF-mediated induction of Cox-2 protein and synthesis of prostaglandin E2. Retinoids also suppressed the induction of Cox-2 mRNA by EGF. Transient transfection experiments showed that EGF caused about a 100% increase in Cox-2 promoter activity, an effect that was suppressed by retinoids. Levels of epidermal growth factor receptor were unaffected by retinoids. Epidermal growth factor caused a nearly 10-fold increase in mitogen-activated protein kinase activity; this effect was not blocked by retinoids.

Topics: Carcinoma, Squamous Cell; Cyclooxygenase 2; Epidermal Growth Factor; Humans; Isoenzymes; Membrane Proteins; Mouth Neoplasms; Prostaglandin-Endoperoxide Synthases; Retinoids; Transcription, Genetic; Tumor Cells, Cultured

Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; G1 Phase; Humans; Keratinocytes; Molecular Sequence Data; Mouth Neoplasms; Proliferating Cell Nuclear Antigen; Resting Phase, Cell Cycle; Time Factors; Tumor Cells, Cultured

Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in the salivary secretion of EGF are of interest because of the importance of EGF in mucosal regeneration. Immunohistochemical studies on normal tissue from parotid and submandibular glands have demonstrated EGF in the serous acini with a tendency to single cell expression in the parotid gland. Amylase has been found in the serous acini of both the submandibular and parotid glands. Haptocorrin was localized in the duct system of both glands. In the submandibular glands with radiotherapy induced sialoadenitis only very few acini with weak or no staining for EGF and amylase were demonstrated, while no changes were observed in the staining for haptocorrin. Analysis on stimulated whole saliva samples collected from 20 healthy individuals and from 20 patients prior to, and 1, 2 and 3 weeks following radiotherapy showed significant reduction in salivary contents of EGF and amylase after treatment as expressed per g protein (p < 0.0002). The salivary content of haptocorrin increased significantly after treatment (p < 0.002). These alterations may be explained by the different cellular sites of the molecules studied, the serous acini being more sensitive to ionising radiation than the duct system. The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p < 0.02), which may indicate that the tumors induce increased secretion of salivary EGF, or alternatively that the oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.

Topics: Aged; Amylases; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Middle Aged; Mouth; Mouth Neoplasms; Nasopharyngeal Neoplasms; Nasopharynx; Salivary Glands

The effect of local administration of epidermal growth factor (EGF) on 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tumour formation was investigated in a hamster cheek pouch carcinogenesis model. DMBA-treated hamsters underwent either sialoadenectomy (groups 1 and 2) or a sham operation (groups 3 and 4). Thereafter, EGF (groups 1 and 3) or vehicle (groups 2 and 4) was applied to the cheek pouches for 6 weeks. Fourteen weeks after the beginning of the experiment, the number of cheek pouch tumours was significantly greater in EGF-treated hamsters than in vehicle-treated hamsters, irrespective of whether the submandibular glands had been removed. The number of forestomach tumours, induced by DMBA application to the cheek pouches, was also increased by EGF. These results suggest that EGF applied from the luminal side of the mucosa stimulates tumour formation in the hamster cheek pouch and forestomach.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Administration, Topical; Animals; Cheek; Cricetinae; Drug Synergism; Epidermal Growth Factor; Male; Mesocricetus; Mouth Mucosa; Mouth Neoplasms; Salivary Glands; Stomach Neoplasms

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor alpha (TGF-alpha) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-alpha than normal cells. There was no statistical correlation between the autocrine production of TGF-alpha, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-alpha were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-alpha to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Keratinocytes; Male; Middle Aged; Mouth Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Suramin, a drug used for the treatment of African trypanosomiasis and onchoceriasis, has potential anti-cancer activity. As to the mechanism of action, it is considered to interfere with the action of tumor growth factor and specific growth enzymes. In the present study, the effect of suramin on proliferation of human head and neck squamous cell carcinoma cells was determined. A significant growth inhibition in all three cell lines tested was observed when the concentration of suramin was 100 micrograms/ml or more. The hypothesis was tested to ascertain whether growth inhibition may be due to selective interference with the action of epidermal growth factor. Suramin at growth inhibiting concentrations only partly antagonized the growth stimulating effect of EGF indicating that other mechanisms of action may also contribute to the growth inhibitory activity of suramin. Furthermore, evidence was found that serum proteins other than growth factors presented in the culture medium have a growth stimulating effect on cells and a strongly antagonistical effect on suramin activity.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Mouth Neoplasms; Pharyngeal Neoplasms; Serum Albumin, Bovine; Suramin; Tumor Cells, Cultured

The effects of removal of the submandibular gland (sialoadenectomy) and administration of human urinary epidermal growth factor on the 9,10-dimethyl-1,2-benzanthracene-induced tumor formation were investigated with the use of a hamster cheek pouch model. Syrian hamsters were treated with 0.5% 9,10-dimethyl-1,2-benzanthracene for 6 weeks. Thereafter hamsters in group 1 underwent a sham operation and those in groups 2 and 3 underwent a sialoadenectomy. Subsequently, hamsters in groups 1 and 2 were given 0.9% sodium chloride and group 3 received the human urinary epidermal growth factor at a dose of 0.25 mg/kg body weight subcutaneously three times a week for 8 weeks. Sixteen weeks after the start of the experiment, the mean number of tumors that were less than 3-mm in diameter in groups 1 and 3 was significantly greater than that in group 2 (p < 0.05). The overall incidence and mean number of all carcinomas irrespective of size showed no differences among the experimental groups. These results indicate that epidermal growth factor synthesized in the submandibular gland may enhance the induction of cheek pouch tumor.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cricetinae; Epidermal Growth Factor; Hyperplasia; Male; Mesocricetus; Mouth Neoplasms; Papilloma; Submandibular Gland

Humoral hypercalcemia of malignancy is a paraneoplastic syndrome associated with a variety of solid neoplasms including squamous cell carcinomas of various sites. Parathyroid hormone-related protein (PTHrP) is a newly recognized hormone that has been implicated as one of the major causative factors in the pathogenesis of this syndrome. A canine oral squamous carcinoma cell line (SCC 2/88) was used to investigate the regulation of production of PTHrP in response to agents that alter keratinocyte differentiation/proliferation in vitro.. SCC 2/88 cells grown in serum-free media were exposed to various factors and PTHrP production was measured by radioimmunoassay. This cell line spontaneously produced substantial amounts of PTHrP (up to 7,000 pg/ml) without the need for a fibroblast-feeder layer. Production of PTHrP decreased at cellular confluence, and with increasing passage number.. Epidermal growth factor, cholera toxin, calcium, 1,25-dihydroxyvitamin D, ionomycin, trans-retinoic acid, transforming growth factor-beta 1 and hydrocortisone stimulated production of PTHrP by SCC 2/88 cells to various degrees. Transforming growth factor-beta 1 was the most potent stimulator of PTHrP production, with a maximal stimulation of 25-fold over control. Monensin decreased PTHrP secretion as early as 6 hours post-treatment and by 48 hours, there was no detectable PTHrP in the conditioned cell culture medium. Calcium, cholera toxin, ionomycin, and transforming growth factor-beta 1 decreased keratinocyte proliferation as measured by cell counts at all doses tested.. The results of this study revealed that SCC 2/88 cells spontaneously produced substantial amounts of PTHrP under baseline conditions and that compounds known to affect keratinocyte differentiation/proliferation up-regulated production of PTHrP. These cells will be valuable to investigate the molecular regulation of PTHrP production by squamous cell carcinomas.

Topics: Animals; Calcitriol; Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cholera Toxin; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Hydrocortisone; Ionomycin; Keratinocytes; Kinetics; Mouth Neoplasms; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Radioimmunoassay; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Exogenous administration of epidermal growth factor (EGF) enhances tumor growth of H2T, a hamster pancreatic cancer that is inoculated in the cheek pouch. The effect of endogenous EGF on tumor growth, however, is not known. The main source of EGF in the body is the submandibular glands. This study examined the influence of submandibular gland resection (SMx) on H2T tumor growth in the cheek pouch and compared it to the growth in SC tissue. Bilateral SMx or sham operation was performed on male Syrian golden hamsters. In 30 hamsters (n = 15 for each operation group), 5 x 10(4) and 5 x 10(5) H2T cells were inoculated in the bilateral cheek pouches and intrascapular SC tissue, respectively (Exp. 1). In another 30 hamsters (n = 15 for each operation group), 5 x 10(5) and 1 x 10(6) H2T cells were inoculated at the same sites (Exp. 2). Two longest perpendicular diameters of the tumor were measured once a week for 12 wk (Exp. 1) or 8 wk (Exp. 2), and the tumor area was calculated. The tumor area in the cheek pouch became significantly smaller in the SMx group than the sham-operated group after the 8th wk (Exp. 1) or the 7th wk (Exp. 2). On the other hand, the tumor area in the SC tissue did not show any difference between groups through the experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cheek; Cricetinae; Epidermal Growth Factor; Male; Mesocricetus; Mouth Neoplasms; Neoplasm Transplantation; Pancreatic Neoplasms; Skin Neoplasms; Submandibular Gland

Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 micrograms/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10,000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [3H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.

Topics: Breast Neoplasms; Cell Survival; Colonic Neoplasms; Cycloheximide; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Leucine; Mouth Neoplasms; Protein Biosynthesis; Tumor Cells, Cultured

Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected with a monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa, the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus paralleling the situation observed in the normal differentiated oral mucosa. In four cases, material was available from both a primary tumour and a metastasis. Three of these were positive for TGF-alpha and EGF with the same staining pattern as that of the primary tumours. This investigation together with our previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Mouth Mucosa; Mouth Neoplasms; Transforming Growth Factor alpha

The A9 antigen is a basement membrane antigen of normal squamous epithelial cells that is strongly expressed in many squamous carcinomas. High expression of this antigen is associated with early relapse in squamous cell carcinomas of the head and neck. We now know that the A9 antigen is structurally, immunologically, and functionally similar to the alpha 6 beta 4 integrin that has been shown to be linked to metastatic behavior in murine tumor models. The alpha 6 and beta 4 genes have been cloned and sequenced, and a model has been constructed from the deduced amino acid composition. In this study we present a hypothetical model and use it to design experiments to assess the factors that influence the expression of the A9/alpha 6 beta 4 integrin in normal and malignant keratinocytes. High calcium induces down regulation of A9/alpha 6 beta 4 antigen in normal but not malignant keratinocytes within 24 hours. Although calcium can down-regulate beta 4 message in tumor cells in the absence of epidermal growth factor (EGF), transcription of beta 4 increased in the tumor cells under the conditions we used for assessing antigen expression (calcium plus EGF). Retinoic acid also stimulated transcription of beta 4 in tumor cells, but this was partially inhibited by the presence of high calcium. Phosphorylation of the beta 4 chain was stimulated by epidermal growth factor and calcium in normal keratinocytes, but in the malignant cells phosphorylation was constant regardless of the culture conditions. Our results indicate that high expression of the alpha 6 beta 4 integrin is associated with conditions that favor migration and undifferentiated proliferation of normal keratinocytes and that malignant keratinocytes differ from normal keratinocytes by constitutive phosphorylation of beta 4 and by failure to downregulate beta 4 transcription in response to calcium in the presence of EGF.

Topics: Antigens, Neoplasm; Blotting, Northern; Calcium; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; Humans; Integrins; Keratinocytes; Models, Molecular; Mouth Neoplasms; Phosphorylation; Proteolipids; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.

Topics: Animals; Carcinoma, Squamous Cell; Cell Nucleus; Epidermal Growth Factor; Humans; Interphase; Liver; Liver Neoplasms, Experimental; Melanoma, Experimental; Mitosis; Mouth Neoplasms; Neoplasms, Experimental; Neutral Red; Rats; Skin Neoplasms; Tumor Cells, Cultured

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Keratinocytes; Mice; Mice, Nude; Mouth Neoplasms; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous

This study examined the characteristics of keratinocytes from 4-nitroquinoline N-oxide-treated rat oral epithelium that formed well differentiated carcinomas or spindle cell tumours when transplanted s.c. to athymic mice. Small polygonal-type cells in early culture passage gave rise to xenografts that were well differentiated carcinomas with keratin positivity and a differential reactivity with two anti-vimentin antibodies (positive Vim Dako, negative Vim 3B4). Progressive culture of the polygonal cells resulted in cells of a more stellate-like morphology which, when transplanted, formed spindle cell tumours that were keratin negative and vimentin positive (Vim Dako and Vim 3B4). The staining pattern of the xenografts was similar to that of the cultured cell derivatives. By contrast to normal oral keratinocytes which were stimulated by epidermal growth factor (EGF), the parental and xenograft-derived cells were markedly less responsive and at times refractory to EGF. Low affinity EGF receptors characterized normal keratinocytes whilst parental and xenograft-derived cells from well differentiated carcinomas and spindle cell tumours expressed diminished numbers of low and high affinity and high affinity EGF receptors respectively. Normal keratinocytes and parental tumour cells were inhibited by transforming growth factor (TGF-beta) whereas xenograft-derived cell lines from both well differentiated carcinomas and spindle cell tumours were refractory to TGF-beta. TGF-beta receptors were predominantly of high affinity although xenograft-derived cells, particularly from spindle cell tumours, expressed increased numbers of receptors which were of lower affinity. The results indicate that spindle cell tumours are a variant of well differentiated carcinomas and express an aberrant pattern of differentiation. Resistance to EGF-induced stimulation and TGF-beta-induced inhibition appears not to be an integral part of the tumour phenotype but the pattern of EGF and TGF-beta receptor expression closely follows the degree of tumour differentiation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Keratinocytes; Male; Mouth Neoplasms; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Thymidine; Transforming Growth Factor beta; Tritium

The immunohistochemical localizations of human epidermal growth factor (hEGF) and EGF receptor (EGFr) in oral tissues, including normal mucosa, leukoplakia and squamous cell carcinoma were examined by the use of monoclonal antibodies to hEGF and EGFr. In normal mucosa and leukoplakia, immunostaining of hEGF was limited to an underlying layer of connective tissue near the epithelium. The intensity of extracellular staining appeared to increase with the degree of epithelial malignancy and was eventually most striking in the stroma of invasive carcinoma. The epithelial cells in normal mucosa, leukoplakia, and squamous cell carcinoma showed negligible immunoreactivity for hEGF. Expression of EGFr appeared to be associated with the proliferative activity of cells and/or epithelial malignancy. In normal mucosa, anti-EGFr monoclonal antibody reacted only with the basal cell layer. In all sections of leukoplakia, the positive cells for EGFr were found in the prickle cell layer in addition to the basal cell layer. Most tumour cells in squamous cell carcinoma were strongly positive for EGFr. These findings indicate increased expression of hEGF and EGFr with malignancy. The characteristic localization of extracellular hEGF in the underlying connective tissue and in stroma of oral mucosal tumours suggests a possible epithelial-mesenchymal interaction in hEGF secretion.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Leukoplakia, Oral; Mouth Mucosa; Mouth Neoplasms

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.

Topics: Animals; Carcinoma, Squamous Cell; Cell Count; Cell Differentiation; Cell Division; Cells, Cultured; Cholera Toxin; Culture Media; Dog Diseases; Dogs; Epidermal Growth Factor; Histocytochemistry; Immunohistochemistry; Keratinocytes; Microscopy, Electron; Microscopy, Electron, Scanning; Mouth; Mouth Neoplasms; Tumor Cells, Cultured

This study examined the characteristics of premalignant oral epithelial cell lines derived from non-invasive palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vivo. In contrast to normal keratinocytes, premalignant epithelial cells had an extended life span, were independent of 3T3 fibroblast support, and expressed variable anchorage independence in gel culture and tumorigenicity in athymic mice. The expression of these functional phenotypes did not correlate with the duration of 4NQO treatment. Keratinocytes from 4NQO-treated tissues predominantly had fewer epidermal growth factor (EGF) receptors than normal controls. The expression of high-affinity EGF receptors paralleled the emergence of the anchorage-independent phenotype and was markedly elevated in tumorigenic cell lines. Cell lines with an extended life span expressed fewer transforming growth factor beta 1 (TGF-beta) receptors than their normal counterparts though the loss of these receptors appeared to be unrelated to either anchorage independence or tumorigenicity. Normal keratinocytes were stimulated and inhibited, in a dose-dependent manner, by EGF and TGF-beta respectively. By contrast, a cell line that was immortal, anchorage dependent and non-tumorigenic showed reduced sensitivity to stimulation by EGF and was inhibited only by high concentrations of TGF-beta. Cells that were immortal, anchorage independent and tumorigenic, however, were refractory to EGF and were inhibited only by high concentrations of TGF-beta. There was no correlation between the expression of EGF or TGF-beta cell surface receptors and the response to ligand binding. The results show that tumour progression in rat oral epithelial cells is associated with a progressive independence of growth factor control. The number and distribution of EGF and TGF-beta receptors may be useful markers in more closely defining the stages of epithelial tumour progression.

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Keratinocytes; Kinetics; Male; Mice; Mice, Nude; Mouth Mucosa; Mouth Neoplasms; Palate; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Tongue; Transforming Growth Factors

Transforming growth factors have a wide range of biological activities related to cell proliferation and differentiation. In general TGF-alpha promotes cell proliferation while TGF-beta may stimulate or inhibit proliferation depending on the cell type and growth factor environment. Cultured human keratinocytes, skin and oral squamous cell carcinomas were analysed for the presence of transcripts and protein for the transforming growth factors alpha & beta. Both growth factors were detected in cultured keratinocytes (which have receptors for and respond to both ligands), and in medium conditioned by these cells. Additionally transcripts for TGF-alpha were found preferentially in the basal, proliferative compartment of cultured keratinocytes. Similarly both growth factors were detected in oral squamous cell carcinomas and a highly significant inverse correlation was found between the levels of TGF-alpha and the epidermal growth factor receptor in these tumours. The data for TGF-alpha are consistent with the existence of an autocrine growth control loop influencing cell proliferation in both a normal cell type and malignant epithelial tissues, a process that in keratinocytes and responsive squamous cell carcinomas could be modulated by TGF-beta.

Topics: Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epithelial Cells; Humans; Keratins; Mouth Neoplasms; Protein Precursors; Skin; Transforming Growth Factors; Tumor Cells, Cultured

Morphometric assessment including epithelial indices which express morphological features of the epithelium, mitotic index, mean nuclear area, mean form factor of the nucleus and cellular infiltration in the stroma was performed in 14 cases with oral non-dysplastic epithelium and 66 cases with dysplastic epithelium. The results from morphometry showed a close relationship to the histological severity of dysplasia determined by the histological criteria of Bánóczy. Immunohistochemical localization of carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and epidermal growth factor (EGF) were investigated in 14 cases of non-dysplastic epithelium, 66 cases of dysplastic epithelium and 16 cases of squamous cell carcinoma, and compared with the morphometric results. Positive rates of CEA and EMA reaction in epithelial dysplasia increased with the advance of the dysplastic grade. Those of EGF reaction decreased with the advance of the dysplastic grade. It is suggested that morphometry and immunohistochemistry are useful in confirmation of the histological severity of oral epithelial dysplasia.

Topics: Antigens, Neoplasm; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Chi-Square Distribution; Epidermal Growth Factor; Humans; Immunohistochemistry; Leukoplakia, Oral; Membrane Glycoproteins; Mitotic Index; Mouth Neoplasms; Mucin-1

The cheek pouch of the Syrian hamster is an excellent tissue for the experimental induction of oral cancer by carcinogenic chemicals. Lysate prepared from a cell line (HCPC-1) derived from one of these hamster oral tumors greatly increased the growth of these oral tumor cells in vitro. We now show that the mitogenic substance, transforming growth factor alpha (TGF-alpha), is present in all of the chemically transformed hamster oral tumors examined (in vitro and in vivo). In no adult normal tissue of the Syrian hamster can we detect expression of TGF-alpha. TGF-alpha could be partly or wholly responsible for the mitogenic activity detected in the lysate of the chemically transformed hamster oral keratinocytes. Both normal and chemically transformed hamster oral keratinocytes express the receptor to epidermal growth factor. The consistent detection of TGF-alpha and epidermal growth factor receptor mRNAs in these hamster oral tumor cells suggests that an autocrine growth mechanism might be operative. This hamster cheek pouch oral cancer model can be used for the molecular analysis of how TGF-alpha and epidermal growth factor receptor might be involved in the malignant transformation of epithelial tissues.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Cricetinae; DNA Replication; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Growth Substances; Keratins; Mouth Neoplasms; Peptides; Transforming Growth Factors

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.

Topics: Amino Acids; Animals; Carcinoma, Squamous Cell; Cattle; Cell Division; Cell Line; Depression, Chemical; Dogs; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Immunosorbent Techniques; Mice; Mouth Neoplasms; Ovarian Neoplasms; Phosphorylation; Phosphotyrosine; Receptors, Cell Surface; Tyrosine

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Humans; Mouth Neoplasms; Proto-Oncogenes; Receptors, Cell Surface; Skin Neoplasms

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.

Topics: Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; KB Cells; Kinetics; Mouth Neoplasms; Receptors, Cell Surface

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Mouth Neoplasms; Oncogenes; Receptors, Cell Surface

The kinetics of association of epidermal growth factor (EGF) and diferric-transferrin (TF) with clathrin-coated membranes of KB cells were examined using anti-clathrin antibody bound to Staphylococcus aureus. The ligands were bound to cells at 4 degrees C and after warming to 30 degrees C for 5 min, both ligands were found concentrated in a membrane fraction immunoadsorbed with anti-clathrin antibody. Fifteen minutes after entry both ligands moved to a non-clathrin-associated compartment. Twenty to 25 min after entry, EGF, but not TF, appeared in a second clathrin-associated compartment. In parallel morphologic experiments, EGF-horseradish peroxidase (EGF-HRP) and TF-HRP were both found at 6 min in coated pits associated with the plasma membrane. At 22 min EGF-HRP, but not TF-HRP, was localized in Golgi-associated coated pits. These studies provide biochemical and morphological evidence suggesting that coated membranes in the Golgi region are involved at some stage in the transfer of EGF to lysosomes. The method should be of general utility in the study of receptor-mediated endocytosis.

Topics: Biological Transport; Carcinoma; Cell Line; Clathrin; Coated Pits, Cell-Membrane; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Microscopy, Electron; Mouth Neoplasms; Receptors, Cell Surface; Receptors, Transferrin; Transferrin

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.

Topics: Antibodies, Monoclonal; Biological Transport; Carcinoma; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; KB Cells; Kinetics; Lysosomes; Microscopy, Electron; Mouth Neoplasms; Receptors, Cell Surface

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.

Topics: Adenoviruses, Human; Amines; Ammonium Chloride; Carcinoma; Cell Line; Chloroquine; Drug Synergism; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Mouth Neoplasms; Protein Biosynthesis; Pseudomonas; Receptors, Virus

When KB cells labeled with 51Cr (1 muCi/ml) were treated with adenovirus type 2 (Ad2) at pH 6, 51Cr was released in a concentration-dependent manner with half-maximal release at 1 microgram/ml of virus. The 51Cr release was maximum at 10 micrograms/ml and represented nearly 20% of cell-associated 51Cr. 51Cr release depended on the length of incubation with Ad2 and the pH of the medium; maximum release was at pH 6 with very little release at pH 7.5. An antiserum against the penton base of Ad2 specifically neutralized the ability of Ad2 to release 51Cr. Chloroquine up to 200 microM did not block Ad2-dependent 51Cr release. DEAE-dextran stimulated Ad2-dependent 51Cr release; 4-5-fold stimulation was observed at 50 micrograms/ml of DEAE-dextran. We have compared the ability of Ad2 to release 51Cr with its ability to disrupt endocytic vesicles. Vesicle disruption was independent of pH of the medium in the range of pH 6 to 7.5 and was blocked by chloroquine, whereas, 51Cr release was much greater at pH 6 than at pH 7.5 and was not affected by chloroquine. These results suggest that Ad2 possess a lytic activity which ordinarily lyses acidic endocytic vesicles but, if the cells are maintained at acidic pH, can also directly disrupt the plasma membrane.

Topics: Adenoviruses, Human; ADP Ribose Transferases; Bacterial Toxins; Carcinoma; Cell Transformation, Viral; Chloroquine; Chromium Radioisotopes; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; KB Cells; Kinetics; Mouth Neoplasms; Pseudomonas aeruginosa Exotoxin A; Virulence Factors

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Mice; Mice, Inbred BALB C; Molecular Weight; Mouth Neoplasms; Receptors, Cell Surface

Topics: Carcinoma; Cell Line; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Freezing; Humans; Kinetics; Macromolecular Substances; Mouth Neoplasms; Phorbols; Receptors, Cell Surface; Tetradecanoylphorbol Acetate