Human adenoidcysticcarcinoma (ACC) is characterized by diffused invasion of the tumor into adjacent organs and early distant metastasis. Anoikisresistanceandepithelialmesenchymaltransition (EMT) are considered prerequisites for cancer cells to metastasize. Exploring the relationship between these processes and their underlying mechanism of action is a promising way to better understand ACC tumors. We initially established anoikis-resistant sublines of ACC cells; the variant cells revealed a mesenchymal phenotype through Slug-mediated EMT-like transformation and displayed enhanced metastaticpotential both in vitro andin vivo. Suppression of EMT by knockdown of Slug significantly impaired anoikisresistance, migration, and invasion of the variant cells. With overexpression of Slug and Twist, we determined that induction of EMT in normal ACC cells could prevent anoikis, albeit partially. These findings strongly suggest that EMT is indispensable inanoikisresistance, at least in ACC cells. Furthermore, we found that the EGFR/PI3K/Akt pathway acts as the common regulator for EMT-like transformation andanoikisresistance, as confirmed by their specific inhibitors. Gefitinib and LY294003 restored the sensibilities ofanoikis-resistant cells to anoikisand simultaneously impaired their metastaticpotential. In addition, the results from our in vivo model of metastasis suggest that pretreatment with gefitinib promotes mouse survival by alleviating pulmonary metastasis. Most importantly, immunohistochemistry of human ACC specimens showed a correlation between the overexpression of Slug and EGFR staining. This study has demonstrated that Slug-mediated EMT- like transformation isrequired by human ACC cells to achieve anoikisresistanceand their metastaticpotential. Targeting the EGFR/PI3K/Akt pathway holds potential as a preventive strategy against distant metastasis of ACC.
Although several studies have investigated the expression of TrkB in solid cancers [6–8], this is the first study to address the function of TrkB in human EC. Anger et al.  showed that TrkB protein is expressed in human endometrium, and its expression may be greater in women with endometriosis. This finding implies that human endometrium has ectopic growth traits. We found that the normal endometrium produces weak levels of TrkB. but that TrkB was extensively detected in EC. A correlation between TrkB and BDNF expression was also found in EC, which isin accordance with reports of other tumors [32,33]. Previous studies demonstrate that TrkB is an independent prognostic factor for ovarian and gastric cancers [23,34]. Consistently, our results revealed that high TrkB levels are associated with lymph node metastasis and lymphovascular space involvement, which are associated with high risk factors in EC patients. Expression of TrkB has also been associated with poor tumor pathological grade or advanced tumor stage in several solid tumor entities including ovarian cancer, lung cancer and colorectal carcinoma [23,35,36], however, our exploratory clinical study could not confirm this association statistically in EC. Nevertheless, even with our limited patient numbers, a trend is observed that the expression of TrkB in EC pathological grade G3 (7/9, 77.8%) is slightly higher than that in G1 (33/45, 73.3%) and G2 (31/40, 77.5%) (Table 1). Furthermore, though no statistical correlation was observed in TrkB expression for the 110 patient samples categorized by histological type, we demonstrated that the mean TrkB level in UPSC and ECCC were higher than in endometrioid adenocarcinoma (Figure 1B). Thus, though TrkB expression is clearly upregulated in EC, correlates with EC- associated risk factors and has a clear role inmetastaticpotential, future studies with larger numbers of patients will be necessary to precisely define the correlation of TrkB expression with the EC grade or histological type. Though these results are not sufficient to definitively designate TrkB as a prognostic factor for EC, our expression data combined with our functional data suggest that TrkB might serve as a target for anticancer therapy.
enhance TWIST1 expression but is more likely to induce its nuclear re-localization that associated with the gain of a mesenchymal phenotype. In this model the loss of CDH1 expression is only an indirect proof but suggests that TWIST1 might directly bind DNA to induce EMT. Post-translational regulation of TWIST1 might also be mediated through mutated EGFR activation, indeed, in head and neck cancer, IL6 activation was recently shown to stabilize TWIST1 through CK2 phosphor- ylation . The mutated EGFR/TWIST1 interdependence was validated using RNA silencing. TWIST1 depletion in other cancers such as prostate and gastric carcinoma also counteract the EMT program . In our model TWIST1 expression also favored cell mobility and could therefore enhance the metastaticpotentialof tumor cells. Other transcription factors have been implicated in the acquisitionof an EMT such as ZEB1, E47, SNAI1, and SNAI2 . In our experience ZEB1 and SNAI1 were slightly increased in H1650 cells after EGF treatment and SNAI2 was up-regulated at protein level suggesting a possible cooperation between transcrip- tion factors. This was not retrieved for the TWIST1 non- expressing EGFR mutated cells in which ZEB1 and SNAI1 Figure 6. TWIST1 depletion through RNA interference overrides EMT induction by EGF in H1650 cells. EGFR mutated, TWIST1 expressed H1650 human lung cells were depleted of TWIST1 through RNA interference. Impact of TWIST1 depletion on epithelialandmesenchymal markers in absence or in presence of EGF was examined. A. The efficiency of the siRNA was controlled by assessing TWIST1 expression by immunofluorescence. B. Expression analysis of TWIST1, CDH2, VIM, CDH1 and JUP in absence of EGF treatment by quantitative RT-PCR. Graph shows the relative expression (siRNA TWIST1/siRNA scramble) for the 5 markers. Each column represents the mean 6SD of 3 independent experiments each one done in triplicate. C. Impact of TWIST1 depletion on TWIST1, CDH2, CDH1 and JUP protein expression in H1650 cells in absence or in presence of EGF as assessed by western blotting. D. Impact of TWIST1 depletion on TWIST1, CDH2, VIM, CDH1 and VIM following cell treatment with EGF as assessed by relative expression (siRNA TWIST1/siRNA scramble). Each column represents the mean 6SD of 3 independent experiments each one done in triplicate. doi:10.1371/journal.pone.0029954.g006
after external beam irradiation in 1 case of inoperable sq- uamous carcinomaof the lower third of the trachea, which was asymptomatic at that time. The other patient with sq- uamous cell carcinoma was previously irradiated for a head and neck tumor and received brachytherapy alone. He had an inoperable tumor located at the upper portion of the trachea, causing dyspnea, cough, and mild bleed- ing right below the supraclavicular radiation field limits. The 2 patients with adenoidcysticcarcinomaand plas- macytoma, respectively, underwent brachytherapy as a sal- vage treatment for local recurrences after external beam irradiation years before (9 and 11 years, respectively). Di- agnosis of recurrence was made after complaints of dyspnea and cough with mild bleeding in the first case and cough in the second one. Only the patient with plasmacytoma received laser therapy for debulking before irradiation. 14
Adenoidcysticcarcinoma was the most prevalent malignant SGT (14 cases), representing 12.8% of all tumors and 58.3% of malignant tumors, followed by mucoepidermoid carcinomaand acinic cell carcinoma. The predominance ofadenoidcysticcarcinoma has been described in studies conducted in Congo, Tanzania, Croatia and Nigeria. In contrast, studies conducted in Iran, Brazil, Jordan, the USA, the United Kingdom and Italy report a greater frequency of mucoepidermoid carcinoma than adenoidcysticcarcinoma, whereas similar distribution between these two types of SGTs has been reported in China, Finland and Mexico (Table 4). These indings Table 4. Distribution of salivary gland tumors in different countries.
With these limitations in mind, we undertook a literature search of PubMed (including Medline) using the search strategy ‘‘adenoidcysticcarcinoma larynx’’ and ‘‘adenoidcysticcarcinoma head neck’’ which resulted in 1292 articles, which were hand-searched for pertinent articles. Their reference lists were also searched for additional cases. We excluded cases without a definitive diagnosis of AdCC, cases where the location of the tumor was not definitely the larynx and cases which were included in previously published reviews (as stated in the table, see remarks). As stated above only a few cases reported include a complete staging procedure, the extent of neck dissections, histology images illustrating the definitive diagnosis, why the authors performed a neck dissection, follow-up of the patient, recurrence of the lesion and location of the recurrence. Our comprehensive review of AdCC of the larynx reported in the literature from 1912 to 2015 includes 252 cases as shown in Table 1 [3, 5–110] (excluded cases have been summarized in Table 2). Based on these data, our review indicates that AdCC of the larynx occurs in individuals averaging 52.3 years of age (range 12–84 years) andis more common in males (60.7%). The most frequent site of origin is the subglottis (58.2%) followed by the supraglottis (32.1%) and glottis (9.7%). Of the 252 cases, the status of the regional lymph nodes was specifically mentioned in 156, andof these 24 (15.4%) were associated with cervical lymph node metastasis. Due to the lack of sufficiently long term follow-up, the number of 47 cases reported to have had distant metastasis is probably underestimated.
Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression andepithelial-to-mesenchymaltransition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O 2 . The
regulation of vimentin [16,28]. Furthermore, the DNA microarray results highlighted the corresponding molecular features of miR- 122-expressing Sk-hep-1 cells (Figures 1D, E and Table S4 in File S1), strongly supporting the aforementioned biological transfor- mations. Collectively, the results of the present study and those obtained in other studies [16,25,28] fully support that the ectopic expression of miR-122 in liver cancer cells triggers MET that may contribute to the inhibition of motility and invasion of cancer cells. As a cell moves in a designated direction, the cooperation between continuous actin polymerization and depolymerization facilitates the protrusion of the cell at the anterior front end . In addition, the cell undergoes consecutive actomyosin contrac- tions and separates from the posterior end, resulting in directional cell movement . Moreover, the formation of the stress fibers, filopodia and lamellipodia also requires actin polymerization . Figure 5. HNF4a over-expression induced MET-like cellular marker alterations and suppressed cell motility and invasion by up- regulating miR-122. (A) The over-expression of HNF4a induced an epithelial-like phenotype. Phage contrast images of the morphology of Bel-7402 cells expressing either the control vector or HNF4a are shown (original magnification: 6200). (B) The ectopic expression of HNF4a triggered MET-like cellular marker alterations through the up-regulation of miR-122. The cell extracts from Bel-7402 cells carrying the control vector, HNF4a or HNF4a and miR-122 inhibitor were analyzed through Western blotting with antibodies against the indicated proteins. (C) HNF4a over-expression suppressed cell motility and invasion in Bel-7402 cells via up-regulation of miR-122. The motility and invasion of Bel-7402 cells carrying the control vector, HNF4a or HNF4a and miR-122 inhibitor were analyzed using transwell and Matrigel-coated Boyden chambers, respectively. The migrated cells were plotted as the average number of cells per field of view from 3 different experiments, as described in the Materials and methods section.
(interactions 4 and 80) [40,42]. Based on the redundancy of RPTP activity, we represented RPTP kappa and DEP1 by a single node (RPTP). Based on reports that showed RPTP kappa and DEP1 activation is enhanced in high cell confluency, we consider a cell contact activation of RPTP by a generic ligand (RPTPL) [44,45,144]. In this network model, the effects of intracellular phosphatases (e.g. PTPs and PTEN) were neglected assuming that the strength of tyrosine kinase activation is enough to become dominant over intracellular phosphatases [145,146]. This is reasonable to assume in the case of a strong signal to activate tyrosine kinases. We included the transcriptional repression of E-cadherin gene (CDH1) by ZEB and SNAI family of transcription factors (interactions 14 and 15, see also Figure S1 in section 6) [132,147,148]. This was coupled with the core EMT regulatory circuit composed by mutual inhibitions between ZEB/SNAI and miR200 family (interactions 72, 73 and 134) [34,104]. Canonic MAPK and TGF-β signalling were included here because they mediate the activation of SNAI and ZEB, respectively (interactions 110, 116 and 133, see also Figure S1 in section 6) [25,34,80]. We accounted for the main growth factors in the microenvironment ofEpithelial tissues able to drive EMT, the EGF and HGF [25,149,150]. Canonic Wnt signalling was considered because is involved in the autocrine regulation of TGF-β signalling (interaction 130) and participates in the dissolution of E-cadherin/β-catenin complex through CK1 (interaction 81) [31,69,151]. In this pathway, we also included a self-activation on TCF_LEF (interaction 129) to account for an enhanced activation of Wnt signalling due to AKT and MET effects reported incarcinoma cell lines [152,153].
Dental surgeons need to detect any changes in the oral mucosa of their patients. Early diagnosis of ACC results in better quality of life and higher survival rate. The objective of this study was to perform a systematic review of litera- ture on ACC of the head and neck region and its clinical and pathological features, with emphasis on tumor perineural invasion capacity.
Objetivo: Descrever e analisar as características de uma série de casos de portadores de neoplasias epiteliais primárias da glândula lacrimal, o tratamento cirúrgico, assim como os acha- dos histopatológicos. Métodos: Avaliação retrospectiva dos arquivos de pacientes com neoplasias epiteliais primárias da glândula lacrimal, no período de 1997 até 2007. Todos os pacientes com tumores epiteliais primários da glândula lacri- mal foram incluídos neste estudo. Foram analisados os dados sobre sexo, idade, características clínicas, tratamento cirúrgi- co, achados histopatológicos e seguimento dos pacientes. As lâminas com secções histológicas dos tumores foram revisa- das pelo mesmo patologista. Resultados: No período do estu- do, foram encontrados 12 pacientes, sendo 5 (41,7%) portado- res de tumores benignos, todos adenomas pleomórficos (tu- mor benigno misto), e 7 (58,3%) com neoplasias malignas, assim distribuídos: quatro casos de carcinoma adenóide cís- tico, dois de carcinoma mucoepidermóide e um de carcinoma ex-adenoma pleomórfico. Analisando-se de modo global, a idade média dos portadores foi de 54,1 anos (variando de 14 a 70 anos); com média de idade de 52,4 anos (variando de 14 a 65 anos) para neoplasias benignas, e 55,3 para neoplasias malig- nas (variando de 26 a 70 anos). Informações do seguimento, variando de 2 a 10 anos, estavam disponíveis para todos os pacientes. Três pacientes desenvolveram metástases distan- tes e morreram devido à doença. Conclusões: A maioria das neoplasias epiteliais primárias da glândula lacrimal foi o ade- noma pleomórfico e o carcinoma adenóide cístico no período de estudo. Os tumores malignos foram mais frequentes que os benignos. O diagnóstico histopatológico e o estadiamento inicial da doença podem desempenhar uma papel significante na sobrevida do paciente.
EMT is characterized by loss ofepithelial cell polarity, loss of cell-cell contacts, andacquisitionofmesenchymal markers and phenotypic traits that include increased cell motility. E-cadherin is universally expressed by epithelial cells and plays essential roles to form the tight junctions between epithelial cells. Loss of E- cadherin expression is a marker for the occurrence of EMT. We found that after 2 days of TGF-b1 treatment, E-cadherin mRNA levels were steadily decreased in 603B cells as compared with the controls (p ,0.05, Fig. 1B). TGF-b1 treatment did not significantly affect actin mRNA level during the culture (data not shown) and thus, it was used as the internal control to normalize the qRT- PCR data. Western blot analysis revealed a similar but delayed decrease in E-cadherin protein level in TGF-b1-treated cells, comparing with mRNA changes. Specifically, TGF-b1 treatment for 4 days only induced a modest decrease of E-cadherin protein in 603B cells, and a dramatic decrease of E-cadherin protein was detected 6 days after TGF-b1 stimulation (Fig. 1C). Decreased expression of E-cadherin in TGF-b1-treated 603B cells was further confirmed by immunofluorescent staining. As shown in Fig. 1D, whereas cells cultured in the absence of TGF-b1 predominantly expressed E-cadherin on the cell membrane, cells in the presence of TGF-b1 gradually lost E-cadherin expression. In contrast, TGF-b1-treated cells showed an increased expression of N- cadherin, a mesenchymal marker upregulated during EMT . Expression of N-cadherin at both the message and protein levels Figure 1. TGF-b1 induces EMT-associated changes in 603B cells. (A) Morphological alterations of 603B cells induced by TGF-b1 (3 ng/ml) at indicated time points. Upper panel are representative phase images showing the 603B cells cultured in the absence of TGF-b1 and lower panel shows the 603B cells stimulated with TGF-b1. After TGF-b1 exposure, 603B cells gradually assumed a spindle-like shape. (B
Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoidcysticcarcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza- 29deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation- specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.
The human beclin 1 gene is located on chromosome 17q21 (8). The role of autophagy as a tumor suppressor was first broached through genetic studies of beclin 1, and beclin 1 was mapped to a tumor susceptibility locus monoallelically deleted and with a high proportion of human breast, intrahepatic biliary duct, ovarian, and brain cancers. In addition, analysis of human tissue samples revealed that there was a significant loss of beclin 1 protein expression in human breast carcinomas compared to normal breast tissue (9-13). Ectopic beclin 1 expression decreased cancer cell proliferation in vitro and reduced tumorigenic potentialin vivo, further illustrating a role for autophagy in tumor suppression. In support of this concept, two groups illustrated that heterozygous disruption of beclin 1 promoted tumorigenesis in mice, albeit over an extended latency (14,15). These findings demonstrate that
To identify possible regulators that might control the expression of E-cadherin during the EMT, we calculated the regulatory effects of the upstream regulators of E-cadherin. Out of 1732 potential regulators, NetworkProfiler inferred that 370 of them may control the expression of E-cadherin in any of the 762 cancer cell lines (Table S5). These putative regulators were ranked according to the change in their regulatory effect during the EMT. Although we did not include any information on known E-cadherin regulators, about half of the 25 highest ranked regulators were previously reported in the literature (Table 1). For example, 2 zinc finger transcription factors, ZEB1 and ZEB2, are direct repressors of E-cadherin and are involved in the EMT [9,15]. In addition, the miR-200 family indirectly suppresses the EMT by inhibiting the translation of ZEB1 and ZEB2 mRNAs . Similarly, miR-192 inhibits the translation of ZEB2 [13,14]. In addition, SNAI2, a member of the Snail superfamily of zinc finger transcription factors, also is involved in the EMT . Likewise, TCF4 (also known as E2-2), a class I bHLH transcription factor, is an EMT regulator; its isoforms induce the EMT in MDCK kidney epithelial cells . In contrast, FOXA1 and FOXA2 are positive regulators of E-cadherin, which suppress the EMT in pancreatic ductal adenocarcinoma . KLF4 also inhibits the EMT by regulating E-cadherin expression . NetworkProfiler also identified several other known direct repressors of E-cadherin, such as TWIST1  and TCF3 (also known as E47) ; however, these regulators were ranked 38th and 84th, respectively.
targets. Outside the cell, HMGB1 is a potent mediator of inflammation that binds to the receptor for advanced glycation end products (RAGE) with high affinity (4). It causes an acute inflammatory response with accumulation of neutrophils in the interstitial and intra-alveolar areas and production of proinflammatory cytokines in the lung (5). HMGB1 is upregulated in experimental pulmonary fibrosis, particularly during the phase of acute exacerbation, which suggests HMGB1 can be a therapeutic target for IPF (6,7). Pulmonary rehabilitation mixture (PRM) comprises eight traditional Chinese medicines, including Astragalus membra- naceus (Fisch) Bge., Codonopsis pilosula (Franch.) Nannf., Ophiopogon japonicas, Schisandra chinensis, Panax no- toginseng (Burk.) F. H. Chen., Bulbus fritillariae thunbergii, Rhizoma anemarrhenae, and Glycyrrhiza uralensis. Phar- macological activity of PRM has not yet been reported in experimental pulmonary fibrosis. We therefore investigated the effects of PRM on experimental pulmonary fibrosis in vitro andin vivo, and propose a mechanism of action.
angiogenesis through blocking a number of receptor tyrosine kinases [10,11,22]. However, in addition to the established functions in clinical trials, sorafenib likely has much broader effects than is currently known. The present study sheds light on the novel capacity of sorafenib to reverse coordinated epigenetic switching at both the global level and the local level of critical EMT-associated genes that have been previously shown to play crucial roles during EMT, providing a possible epigenetic mechanism for its clinical benefits in patients with cancer metastasis, organ fibrosis and other EMT-related diseases. Using A549 carcinomaepithelial cells as an in vitro model, we found that treatment with sorafenib in during TGF-b1-induced EMT resulted in a loss of active histone markers (H3K4me3 and/or H3K9ac) at the promoters of TGF-b1, Smad2/3 and downstream EMT mediators such as Snail and Slug (Figure 3). Combined with our previous finding that sorafenib disrupts the phosphorylation of Smad2/3 and STAT3 , we suggest that sorafenib interferes with TGF-b1-induced EMT via a dual mechanism–first via the direct inhibition of targeted kinase phosphorylation and then via transcriptional repression of EMT-related genes by epigenetic regulation. In addition to TGF-b signaling, we found that sorafenib inhibits the coordinated epigenetic switching of a broad range of signaling pathways that have been attributed to many oncogenic disorders, including Ras/Raf/MAPK and ErbB signal transduction (Dataset S1). Earlier studies on the resistanceand sensitivity to sorafenib investigated gene-drug correlations by
development of liver cancer. However, several studies have shown that HBV may replicate in other extrahepatic cells  . Some studies revealed that the virus has extensive reservoirs of extrahepatic replication [5b] . HBV proteins and nucleic acids have been found in a number of non-hepatic tissues including lymph nodes, spleen, bonemarrow, kidney, colon, stomach, periadrenal ganglia, skin, thyroid, pancreas, testis, ovaries, brain, heart and lung tissue  . Other studies have failed to prove the presence of extrahepatic HVB, making controversial results in this context. We tried to analyze the presence of Hepatitis B Virus infection in women with epithelial ovarian carcinoma attending to oncology department of Ibn Rochd University hospital.
Endothelial cells were lysed in buffer [50 mM Tris-HCl, pH 7.4, 1% Tween 20 and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc., Rockford, IL, USA)] for 2 h at 4 ˚ C. The homogenates were spun at 18,000 g for 5 min, and the supernatant was collected. Proteins were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Equal amounts of protein extracts (20 mg) were subjected to 10% SDS-PAGE and blotted onto a PVDF membrane (Merck Millipore, Massachusetts, USA). After blocking, the membrane was incubated with the primary antibody (rabbit anti- syndecan-4 1: 1000; rabbit anti-integrin b5 1: 1000, rabbit anti-HPA1 1: 1000 and mouse anti-GAPDH 1:500) diluted in blocking buffer overnight at 4 ˚ C and followed by incubation with secondary antibodies (1:10000) for 1 h at room temperature. The samples were detected by enhanced chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA). An Alliance mini photodocumentation system from UVITEC (Cambrige, UK) was used to scan the films, and the UVIBAND MAX v1503b software (UVITEC - Cambrige, UK) was used to measure the amount of protein detected by each antibody. The experiments were performed in duplicates and repeated twice.
A denoid cysticcarcinomais a rare tumor originating from the salivary glands, especially when arise the external auditory canal. This tumor has high rate of perineural invasion and metastasis, then must be treated with aggressive surgery combined with postoperative radiation. We report a case of an adenoidcysticcarcinoma arising the external auditory canal of 77 years old female patient, who complained hypoacusis and pain. She was treated by radical mastoidectomy and radiotherapy