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P53 family members with a transactivation domain induce cell cycle arrest and promoteapoptosis. However, ΔNp63 isotypes lacking the transactivation (TA)- domain promote cellproliferation and tumorigenesis in vitro and in vivo. Although p53, TAp63 or TAp73 are stabilizedupon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease andphosphorylation of ΔNp63α in squamous cell carcinoma cells. Further work revealed that RACK1physically associated with the p63α C-terminal domain through its WD40 domain. However,stratifin binds with phosphorylated ΔNp63α in response to cisplatin. Upon DNA damage inducedby cisplatin, stratifin mediated a nuclear export of ΔNp63α into cytoplasm and then RACK1targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase.Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and proteindegradation of ΔNp63α, respectively. Our data suggest that modification and down regulation ofΔNp63α is one of the major determinants of the cellular response to DNA damage in human headand neck cancers.  相似文献   

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Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair and were found to be associated with HNSCC in numerous studies. To establish our overall understanding of possible relationships between DNA repair gene polymorphisms and development of HNSCC, we surveyed the literature on epidemiological studies that assessed potential associations with HNSCC risk in terms of gene–environment interactions, genotype-induced functional defects in enzyme activity and/or protein expression, and the influence of ethnic origin on these associations. We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of HNSCC when DNA repair capacity is reduced.  相似文献   

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The heparin-binding growth factor, MK, promoting tumorigenesis and survival was found to associate with α6β1 integrins. We showed for the first time that MK interacted with TSPAN1 and facilitated the association between TSPAN1 and integrin α6β1 proteins in head and neck squamous cell carcinoma (HNSCC) cells. We found that MK mediated an integrin-dependent tyrosine phosphorylation of FAK and activation of paxillin and Stat1α pathways. As result, downstream target genes implicated in cell migration and invasiveness (e.g. MMP-2 and MMP-26) were overexpressed. We observed that RNAi silencing of the critical signaling intermediates led to decrease of MK-induced migration/invasiveness of HNSCC cells. The major finding of this study is a novel MK-triggered signaling mechanism implicated in migration and invasiveness of HNSCC cells.  相似文献   

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Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98high cells, in contrast to CD98low cells, are able to generate tumors in immunodeficient mice, indicating that CD98high cells have stem cell characteristics. Furthermore, the CD98high subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98low fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.  相似文献   

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Until recently, tumor progression has been considered a multistep process defined by tumor cell mutations and the importance of the surrounding stroma poorly understood. It is now recognized that matrix-degrading enzymes that promote tumor cell invasion are elaborated by both tumor cells and fibroblasts in vivo. To determine the relative role of tumor cell-derived proteases compared with fibroblast-derived proteases, coculture experiments were done with each cell type using an in vitro model of type I collagen degradation. Head and neck squamous cell carcinoma cells in coculture with normal dermal fibroblasts showed matrix degradation, but neither cell type alone produced this effect. Manipulating the in vitro coculture environment showed that collagenolysis in this model was a result of fibroblast-derived matrix metalloproteases (MMP). To explore the possible role of extracellular matrix metalloprotease inducer (EMMPRIN) in this interaction, transfection of EMMPRIN into a cell line with low endogenous EMMPRIN expression was done and showed a significant increase in collagenolysis. Inhibition of collagenolysis with a tissue inhibitor of metalloprotease-2 (TIMP-2) and a synthetic furin inhibitor was observed but not with TIMP-1, which suggested a possible role for membrane type-1 MMP. These results suggest that fibroblast-derived MMPs but not those from tumor cells are important for in vitro collagenolysis and that this process is promoted by tumor cell-expressed EMMPRIN.  相似文献   

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Members of the EGFR/ErbB family of tyrosine kinases are found to be highly expressed and deregulated in many cancers, including head and neck squamous cell carcinoma (HNSCC). The ErbB family, including EGFR, has been demonstrated to play key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSCs) which are believed to be responsible for tumor initiation and maintenance. In this study, we investigated the possible role of EGFR as a regulator of "stemness" in HNSCC cells. Activation of EGFR by the addition of EGF ligand or ectopic expression of EGFR in two established HNSCC cell lines (UMSCC-22B and HN-1) resulted in the induction of CD44, BMI-1, Oct-4, NANOG, CXCR4, and SDF-1. Activation of EGFR also resulted in increased tumorsphere formation, a characteristic ability of cancer stem cells. Conversely, treatment with the EGFR kinase inhibitor, Gefinitib (Iressa), resulted in decreased expression of the aforementioned genes, and loss of tumorsphere-forming ability. Similar trends were observed in a 99.9% CD44 positive stem cell culture derived from a fresh HNSCC tumor, confirming our findings for the cell lines. Additionally, we found that these putative cancer stem cells, when treated with Gefitinib, possessed a lower capacity to invade and became more sensitive to cisplatin-induced death in vitro. These results suggest that EGFR plays critical roles in the survival, maintenance, and function of cancer stem cells. Drugs that target EGFR, perhaps administered in combination with conventional chemotherapy, might be an effective treatment for HNSCC.  相似文献   

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The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC50 of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.  相似文献   

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Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.  相似文献   

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《Free radical research》2013,47(8):913-924
Abstract

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radical (HO?), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO? than with H2O2. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H2O2 or HO? stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.  相似文献   

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The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC), have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.  相似文献   

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BACKGROUND: Catumaxomab is an antibody that binds with one arm epithelial cell adhesion molecule (EpCAM) positive tumors and with the other arm CD3+ T cells. Intravenous application of therapeutic antibodies may result in intravascular cytokine release. AIM: In this pilot trial we assessed whether cytokine release can be controlled by ex vivo cell opsonization and cytokine wash-out before administration of catumaxomab, preserving its anti-cancer activity. In addition, preliminary data on safety of and clinical response to catumaxomab coated autologous immune cells were acquired. METHODS: Peripheral blood mononuclear cells (PBMNC) of four patients with recurrent head and neck carcinoma were collected by leukapheresis, incubated ex vivo with catumaxomab for 24 h and cleared from released cytokines. Each patient received an escalated number of antibody-coated PBMNC equivalent to 1 x 10(4), 1 x 10(5), 1 x 10(6) and 1 x 10(7) CD3(+) cells/kgBW intravenously at bi-weekly intervals. RESULTS: After opsonization, PBMNC released substantial amounts of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in vitro, which were removed before administration. Catumaxomab up-regulated CD25, CD69, and CD83 on PBMNC, and catumaxomab loaded PBMNC released IFNgamma and granzyme B when coincubated with EpCAM(+) BHY cells, suggesting cell activation and target directed biological activity. During the study period, one patient died of aspiration pneumonia and one patient needed a tracheotomy. Treatment related adverse events (AE) occurred at the highest cell dose in two patients, whereas 1 x 10(6) loaded CD3(+) cells/kgBW were well tolerated by all patients. One patient showed stable disease for 6 months and one patient is in complete remission for 27 months. CONCLUSION: Ex vivo opsonization of PBMNC with catumaxomab provided biologically active, tumor targeting cells. Extracorporeal PBMNC coating may be an option to control intravascular cytokine release induced by therapeutic antibodies.  相似文献   

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Background aimsAdoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.MethodsIn a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr51 release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8+ and CD4+ subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.ConclusionThe procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.  相似文献   

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Human papilloma virus (HPV) infection represents an emerging risk factor in head and neck squamous cell carcinoma (HNSCC). In contrast to HPV-negative HNSCC, most cases of HPV-positive HNSCC encode wild-type p53, although the p53 protein in these cells is rapidly degraded via HPV E6-mediated ubiquitination and subsequent proteasomal degradation. This unique feature of HPV-positive HNSCC has raised hope that liberation of wild-type p53 from the E6 protein may have therapeutic benefit in this disease. Indeed, suppression of E6 expression promotes apoptosis in HPV-positive HNSCC cell lines. However, the role of p53 in mediating this cell death has not been determined. Here, we demonstrate that siRNAs targeting the E6/E7 RNA, or treatment with the proteasome inhibitor bortezomib, resulted in upregulation of functional p53 and p53 gene targets in three HPV-positive HNSCC cell lines, but not in HPV-negative HNSCC cells. Apoptosis induced by E6/E7 siRNA in HPV-positive cells was found to be dependent on p53, while bortezomib-induced cell death was modestly p53-dependent. Treatment with subtoxic doses of bortezomib led to cell cycle arrest in HPV-positive, but not HPV-negative HNSCC cells. Moreover, this cell cycle arrest was mediated by p53 and the cell cycle inhibitor p21, the product of a p53 target gene. Collectively, these findings establish that wild-type p53 encoded by HPV-positive HNSCC cells, once liberated from HPV E6, can play important roles in promoting apoptosis and cell cycle arrest.  相似文献   

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Human head and neck squamous cell carcinoma (HNSCC) cultures were established from cancers of two patients. These cells were used to study if phosphorylation reactions by protein kinase A (PKA) and dephosphorylation reactions by protein phosphatases-1 and -2A (PP-1/2A) regulate tumor motility and adhesion to extracellular matrix components, and if this might be associated with cytoskeletal reorganization. Both cultures were motile and adherent to collagen I, fibronectin, vitronectin and laminin. Motility and adhesiveness was dependent on production of prostaglandin E2 PGE2 and on PKA activation. Blocking PP-1/2A activity with okadaic acid resulted in a PKA-dependent increase in m otility and, in some instances, adhesiveness by the HNSCC cells. The okadaic acid-induced increase in motility and adhesiveness coincided with a reduction in filamentous actin. These data suggest PKA and PP-1/2A have opposing effects in regulating the motility, adherence, and actin polymerization.  相似文献   

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Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V+H), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V+H decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V+H. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V+H. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.  相似文献   

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