Expression of truncated products at the 5'-terminal region of RIPK2 and evolutive aspects that support their biological importance.

Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance.

Overexpression of Antiangiogenic Vascular Endothelial Growth Factor Isoform and Splicing Regulatory Factors in Oral, Laryngeal and Pharyngeal Squamous Cell Carcinomas

Background: Overexpression of proangiogenic vascular endothelial growth factor A family VEGFAxxx is associated with tumor growth and metastasis. The role of the alternatively spliced antiangiogenic family VEGFAxxxb is poorly investigated in head and neck squamous cell carcinomas (HNSCCs). The antiangiogenic isoform binds to bevacizumab and its expression level could influence the treatment response and progression-free survival. In this study, the relative expression of VEGFAxxx and VEGFA165b isoforms and splicing regulatory factors genes was investigated in a series of HNSCCs. Methods: VEGFAxxx, VEGFA165b, SRSF6, SRSF5, SRSF1 and SRPK1 gene expression was quantified by quantitative real time PCR in 53 tissue samples obtained by surgery from HNSCC patients. Protein expression was evaluated by immunohistochemistry. Results: VEGFAxxx and VEGFA165b were overexpressed in HNSCCs. Elevated protein expression was also confirmed. However, VEGFA isoforms demonstrated differential expression according to anatomical sites. VEGFAxxx was overexpressed in pharyngeal tumors while the VEGFA165b isoform was up-regulated in oral tumors. The VEGFA165b isoform was also positively correlated with expression of the splicing regulatory genes SRSF1, SRSF6 and SRSF5. Conclusions: We concluded that VEGFAxxx and VEGFA165b isoforms are overexpressed in HNSCCs and the splicing regulatory factors SRSF1, SRSF6, SRSF5 and SRPK1 may contribute to alternative splicing of the VEGFA gene. The findings for the differential expression of the antiangiogenic isoform in HNSCCs could facilitate effective therapeutic strategies for the management of these tumors.

Low frequency of cigarette smoking and the risk of head and neck cancer in the INHANCE consortium pooled analysis.

BACKGROUND: Cigarette smoking is a major risk factor for head and neck cancer (HNC). To our knowledge, low cigarette smoking (<10 cigarettes per day) has not been extensively investigated in fine categories or among never alcohol drinkers. METHODS: We conducted a pooled analysis of individual participant data from 23 independent case-control studies including 19 660 HNC cases and 25 566 controls. After exclusion of subjects using other tobacco products including cigars, pipes, snuffed or chewed tobacco and straw cigarettes (tobacco product used in Brazil), as well as subjects smoking more than 10 cigarettes per day, 4093 HNC cases and 13 416 controls were included in the analysis. The lifetime average frequency of cigarette consumption was categorized as follows: never cigarette users, >0-3, >3-5, >5-10 cigarettes per day. RESULTS: Smoking >0-3 cigarettes per day was associated with a 50% increased risk of HNC in the study population [odds ratio (OR) = 1.52, 95% confidence interval (CI): (1.21, 1.90). Smoking >3-5 cigarettes per day was associated in each subgroup from OR = 2.01 (95% CI: 1.22, 3.31) among never alcohol drinkers to OR = 2.74 (95% CI: 2.01, 3.74) among women and in each cancer site, particularly laryngeal cancer (OR = 3.48, 95% CI: 2.40, 5.05). However, the observed increased risk of HNC for low smoking frequency was not found among smokers with smoking duration shorter than 20 years. CONCLUSION: Our results suggest a public health message that low frequency of cigarette consumption contributes to the development of HNC. However, smoking duration seems to play at least an equal or a stronger role in the development of HNC.

Proteomic approaches identify members of cofilin pathway involved in oral tumorigenesis.

The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the "more-aggressive" group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the "less-aggressive" group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.

Prognostic significance of NDRG1 expression in oral and oropharyngeal squamous cell carcinoma.

Human N-myc downstream-regulated gene 1 (NDRG1) is a metastasis suppressor gene with several potential functions, including cell differentiation, cell cycle regulation and response to hormones, nickel and stress. The purpose of this study was to investigate the immunoexpression of NDRG1 in oral and oropharyngeal squamous cell carcinomas searching for its role in the clinical course of these tumors. We investigated immunohistochemical expression of NDRG1 protein in 412 tissue microarray cores of tumor samples from 103 patients with oral and oropharyngeal squamous cell carcinomas and in 110 paraffin-embedded surgical margin sections. The results showed NDRG1 up-regulation in 101/103 (98.1 %) tumor samples, but no expression in any normal tissue sample. Western blot assays confirmed the immunohistochemical findings, suggesting that lower levels of NDRG1 are associated with a high mortality rate. NDRG1 overexpression was related to long-term specific survival (HR = 0.38; p = 0.009), whereas the presence of lymph-node metastasis showed the opposite association with survival (HR = 2.45; p = 0.013). Our findings reinforce the idea that NDRG1 plays a metastasis suppressor role in oral and oropharyngeal squamous cell carcinomas and may be a useful marker for these tumors.

Total thyroidectomy with ultrasonic scalpel: a multicenter, randomized controlled trial.

BACKGROUND: The ultrasonic scalpel is a recently introduced device in head and neck surgery. Total thyroidectomy is the most common endocrine procedure performed by surgeons. METHODS: This was an open, phase IV, multicenter, randomized controlled trial (RCT) that compared the use of an ultrasonic scalpel with a conventional technique in patients who underwent total thyroidectomy. The outcomes were surgical complication rate, operative time, drainage volume, postoperative pain, and costs. RESULTS: In all, 261 patients were included in 11 centers. There was a mean difference of 17% of operative time in favor of the ultrasonic scalpel group. There were no differences in postoperative complications. There was a difference in costs of 14% in favor of the ultrasonic scalpel group, but it was not statistically significant. CONCLUSIONS: The use of an ultrasonic scalpel was as safe as that of the conventional technique and had the advantage of a shorter operative time and lower postoperative drainage. Costs were not different between groups.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma.

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Genomics and proteomics approaches to the study of cancer-stroma interactions.

BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR > or = | 1.0 |, p< or =0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.

Methylation profile of genes CDKN2A (p14 and p16), DAPK1, CDH1, and ADAM23 in head and neck cancer.

Hypermethylation in the promoter region has been associated with a loss of gene function that may give a selective advantage to neoplastic cells. In this study, the methylation pattern of genes CDKN2A (alias p14, p14(ARF), p16, p16(INK4a)), DAPK1, CDH1, and ADAM23 was analyzed in 43 samples of head and neck tumors using methylation-specific polymerase chain reaction. In the oropharynx, there was a statistically significant association between hypermethylation of the DAPK1 gene and the occurrence of lymph node metastases, and in the larynx there was statistically significant evidence of an association between hypermethylation of the ADAM23 gene and advanced stages of the tumors. Thus, a correlation was observed between hypermethylation of the promoter region of genes DAPK1 and ADAM23 and the progression of head and neck cancer.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Laringectomia near-total: relato de caso / Near total laryngectomy: case report

O objetivo deste estudo foi verificar a evolução clínica fonoaudiológica nos aspectos de deglutição e fonoarticulação de um paciente do sexo masculino, com idade de 56 anos, submetido à Laringectomia Near Total(LNT) associada à operação composta. A avaliação fonoaudiológica realizada no 15º pós-operatório revelou: diminuição dos movimentos de língua, de bochechas e de mandíbula, ressecção do palato mole à direita, qualidade vocal rugosa-molhada nível 2, soprosa 2, tensa 2 e instável 3, pitch adequado, loudness reduzida e inteligibilidade de fala muito comprometida. O paciente apresentava disfagia nível 3, classificada de acordo com a escala de severidade, fazendo uso da sonda nasoenteral. Iniciou tratamento fonoaudiológico para o restabelecimento da alimentação via oral e melhora da fonoarticulação. Após fonoterapia, a deglutição do paciente se tornou funcional (nível 6), com a retirada da sonda nasoenteral. Houve redução do comprometimento da inteligibilidade de fala e da qualidade vocal. A loudness e o pitch encontravam-se adequados. A média da freqüência fundamental para a vogal /a/ foi de 81,89Hz, para vogal /i/, 92,86Hz e para vogal /u/, 262,90 Hz. A fonoterapia contribuiu para a melhora da comunicação oral e da deglutição do paciente, porém o tempo de reabilitação foi prolongado devido à intercorrências durante o processo terapêutico.

Analysis of human papillomavirus prevalence and TP53 polymorphism in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma is a disease associated with tobacco and alcohol abuse. There is evidence that the oncogenic human papillomavirus (HPV) may also be a risk for upper aerodigestive tract cancers. High-risk HPVs encode two early proteins, E6 and E7, that can bind to p53 and pRb, respectively, and induce its degradation or inactivation. The TP53 gene has a single polymorphism at codon 72 of exon 4 that encodes either arginine (Arg) or proline (Pro). The purpose of this study was to evaluate the role of HPV infection and TP53 polymorphism in head and neck cancer. We analyzed 50 tumors, as well swabs of oral mucosa from 142 control individuals, with a polymerase chain reaction technique. The prevalence of HPV in controls was 10.6% and in cancer specimens 16%. The frequency distribution of genotypes in controls was 50% Arg/Arg, 43% Arg/Pro and 7% Pro/Pro; in tumors, it was 52% Arg/Arg, 32% Arg/Pro, and 16% Pro/Pro. Contrary to the results of some studies on cervical cancer, no association between any TP53 genotype or allele and the development of head and neck cancer was observed, regardless of HPV status, except for the Pro/Pro genotype, which is associated with the absence of HPV. The arginine allele appears to protect against head and neck cancers. Also, the data showed that HPV infection results in no increased risk of developing head and neck tumors.

Análise acústica da voz captada na faringe próximo à fonte glótica através de microfone acoplado ao fibrolaringoscópio / Acoustic analysis of voice captured in the pharynx above the glottic source through a microphone on a laryngo-fiberscope

Objetivo: O objetivo deste trabalho foi estudar a voz próximo à sua fonte produtora, as pregas vocais, através de um microfone miniaturizado de aparelho auditivo que foi adaptado para ser acoplado à extremidade de um fibrolaringoscópio, permitindo a captação da voz durante a laringoscopia direta. Forma de estudo: Experimental. Material e Método: A voz foi estudada em um grupo de 50 indivíduos, 25 homens e 25 mulheres sem doenças, através de um programa de análise acústica MDVP (Multi-Dimensional Voice Program) do laboratório de voz Computerized Speech Lab, Model 4300B, da Kay Elemetrics. Amostras de vogais sustentadas /a/, /i/ e /u/ foram captadas de três formas diferentes, primeiramente com um microfone comum externo a 15 cm da boca, em segundo lugar com o microfone especial na faringe a 1,5 cm acima das pregas vocais e por último com o microfone especial externamente a 2 cm da boca. Doze parâmetros acústicos relacionados a freqüência fundamental, amplitude e ruído de cada uma das vogais foram comparadas estatisticamente conforme à sua forma de captação. Resultados: Os resultados mostraram diferenças estatisticamente significativas entre a voz captada pelo microfone comum externo e o microfone especial, em relação à freqüência fundamental, aos parâmetros de variação de periodicidade de freqüência, amplitude e ruído. Conclusão: A diferença do som da fonte glótica do som da voz externa pode mostrar as modificações sofridas pela voz no decorrer da passagem pelo trato vocal.

Estudo da anatomia do ramo mandibular do nervo facial / Anatomic study of the mandibular ramus of the facial nerve

O ramo mandibular do nervo facial inerva os músculos depressor da rima bucal e depressor do lábio inferior. Procedimentos que abordam a regiao submandibular podem lesá-lo, o que leva ao desvio da rima labial e conseqüente deformaçao da estética facial. Neste estudo foram analisados quarenta ramos mandibulares do nervo facial em vinte cadáveres dissecados, demonstrando sua freqüente representaçao por mais de um ramo, a presença de anastomoses com outros ramos e sua distância de até treze milímetros da borda inferior da mandíbula.