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OBJECTIVES: To isolate cancer stem cells (CSC) from a metastatic oral squamous cell carcinoma (OSCC) cell line and investigate their in vitro and in vivo phenotypic characteristics. MATERIALS AND METHODS: Subpopulations with individual staining intensities for CD44 and CD326 were isolated from the OSCC cell line LN-1A by FACS: CD44Low/CD326- (CSC-M1), CD44Low/CD326High (CSC-E), and CD44High/CD326- (CSC-M2). Proliferation, clonogenic potential, adhesion, migration, epithelial-mesenchymal transition markers, and sensitivity to cisplatin and TVB-3166 were analyzed in vitro. Tumor formation and metastasis were assessed by subcutaneous and orthotopic inoculations into BALB/c mice. RESULTS: E-cadherin levels were higher in CSC-E cells while vimentin and Slug more produced by CSC-M2 cells. CSC-M1 and CSC-M2 subpopulations showed higher proliferation, produced more colonies, and have stronger adhesion to the extracellular matrix. All cell lines established tumors; however, CSC-E and CSC-M2 formed larger masses and produced more metastases. CONCLUSION: The CSC subpopulations here described show increased cancer capabilities in vitro, tumorigenic and metastatic potential in vivo, and may be exploited in the search for novel therapeutic targets for OSCC.
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Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell- and SCC-9 cell-derived EVs. A multi-omics integration identified 11 'hub proteins' significantly decreased at the metastatic site compared with primary tumor-derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven 'hub proteins' in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC.
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Vesículas Extracelulares/metabolismo , Neoplasias Bucais/metabolismo , Animais , Linhagem Celular , Humanos , Metabolômica , Camundongos , MicroRNAs , Neoplasias Bucais/genética , Prognóstico , ProteômicaRESUMO
OBJECTIVES: To investigate the potential effect of fatty acid synthase (FASN) inhibitor orlistat to enhance the effectiveness of chemotherapy drugs widely used to treat oral squamous cell carcinomas (OSCC), such as 5-fluorouracil, cisplatin, and paclitaxel. METHODS: The OSCC SCC-9 LN-1 metastatic cell line, which expresses high levels of FASN, was used for drug combination experiments. Cell viability was analyzed by crystal violet staining and automatic cell counting. Apoptosis and cell cycle were analyzed by flow cytometry with Annexin-V/7-AAD and propidium iodide staining, respectively. Cyclin B1, Cdc25C, Cdk1, FASN, and ERBB2 levels were assessed by Western blotting. Finally, cell scratch and transwell assays were performed to assess cell migration and invasion. RESULTS: Inhibition of FASN with orlistat sensitized SCC-9 LN-1 cells to the cytotoxic effects of paclitaxel and cisplatin, but not 5-fluorouracil, which was accompanied by a significant reduction in cyclin B1. The suppression of proliferation, migration, and invasion of SCC-9 LN-1 cells induced by orlistat plus cisplatin or paclitaxel was not superior to the effects of chemotherapy drugs alone. CONCLUSION: Our results suggest that orlistat enhances the chemosensitivity of SCC-9 LN-1 cells to cisplatin and paclitaxel by downregulating cyclin B1.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Cisplatino/farmacologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Orlistate/farmacologia , Orlistate/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ciclina B1/farmacologia , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintases/farmacologia , Neoplasias Bucais/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Fluoruracila/farmacologia , Linhagem Celular Tumoral , Apoptose , Proliferação de Células , Ácido Graxo Sintase Tipo IRESUMO
BACKGROUND: Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. SUBJECTS AND METHODS: Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. RESULTS: Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p < .0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine™ database confirmed the underexpression in OSCC compared with normal tissues (p = .014). CONCLUSIONS: In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/genética , Humanos , Neoplasias Bucais/patologia , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço , Trofoblastos/metabolismo , Trofoblastos/patologiaRESUMO
Stanniocalcin 2 (STC2), a glycoprotein that regulates calcium and phosphate homeostasis during mineral metabolism, appears to display multiple roles in tumorigenesis and cancer progression. This study aimed to access the prognostic value of STC2 in oral squamous cell carcinoma (OSCC) and its implications in oral tumorigenesis. STC2 expression was examined in 2 independent cohorts of OSCC tissues by immunohistochemistry. A loss-of-function strategy using shRNA targeting STC2 was employed to investigate STC2 in vitro effects on proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT) and possible activation of signaling pathways. Moreover, STC2 effects were assessed in vivo in a xenograft mouse cancer model. High expression of STC2 was significantly associated with poor disease-specific survival (HR: 2.67, 95% CI: 1.37-5.21, p = 0.001) and high rate of recurrence with a hazard ratio of 2.80 (95% CI: 1.07-5.71, p = 0.03). In vitro downregulation of STC2 expression in OSCC cells attenuated proliferation, migration and invasiveness while increased apoptotic rates. In addition, the STC2 downregulation controlled EMT phenotype of OSCC cells, with regulation on E-cadherin, vimentin, Snail1, Twist and Zeb2. The reactivation of STC2 was observed in the STC2 knockdown cells in the in vivo xenograft model, and no influence on tumor growth was observed. Modulation of STC2 expression levels did not alter consistently the phosphorylation status of CREB, ERK, JNK, p38, p70 S6K, STAT3, STAT5A/B and AKT. Our findings suggest that STC2 overexpression is an independent marker of OSCC outcome and may contribute to tumor progression via regulation of proliferation, survival and invasiveness of OSCC cells.
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Carcinoma de Células Escamosas/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Bucais/metabolismo , Recidiva Local de Neoplasia/metabolismo , Adulto , Idoso , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/genéticaRESUMO
OBJECTIVE: To analyze the gene and immunohistochemical expression of HIF-1α, GLUT-1, FASN, and adipophilin in normal salivary gland (NSG), pleomorphic adenoma (PA), and carcinoma ex pleomorphic adenoma (CXPA) samples. MATERIAL AND METHODS: The gene expression was investigated by the real-time PCR (qRT-PCR) method in 9 samples of frozen tissues of normal salivary gland, 13 PA, and 10 CXPA. We validated the reactions by immunohistochemistry on 20 samples from NSG, 85 PA, and 44 CXPA. RESULTS: Our results showed that there was no statistically significant difference in HIF-1α gene and immunohistochemistry expression among the tissues studied while FASN gene and immunohistochemistry expression increased along the carcinogenesis of the PA. GLUT-1 was significantly more expressed in tumor tissues (PA and CXPA), although protein is mainly expressed in transformed cells than in PA and NSG. In contrast, adipophilin was significantly more expressed in NSG while the expression of the protein increased in PA and CXPA. CONCLUSIONS: In summary, the data presented here suggest that neoplastic cells reprogram the expression of GLUT-1 and adipophilin to adapt to the tumor microenvironment and reinforce, through immunohistochemical results, a possible transcriptional and post-translational regulatory mechanisms that act on the expression of these genes.
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Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.
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Cerulenina/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Lactonas/farmacologia , Vasos Linfáticos/efeitos dos fármacos , Melanoma Experimental/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Inibidores da Síntese de Ácidos Graxos/farmacologia , Humanos , Linfangiogênese/efeitos dos fármacos , Metástase Linfática , Vasos Linfáticos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Orlistate , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismoRESUMO
An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-ß1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.
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Carcinoma de Células Escamosas/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Intervalo Livre de Doença , Matriz Extracelular/patologia , Feminino , Fibroblastos/patologia , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Neoplasias Bucais/patologia , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Pró-Colágeno/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral/fisiologia , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: ADAM17 is one of the main sheddases of the cells and it is responsible for the cleavage and the release of ectodomains of important signaling molecules, such as EGFR ligands. Despite the known crosstalk between ADAM17 and EGFR, which has been considered a promising targeted therapy in oral squamous cell carcinoma (OSCC), the role of ADAM17 in OSCC development is not clear. METHOD: In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro. In addition, the tumor size, tumor proliferative activity, tumor collagenase activity and MS-based proteomics of tumor tissues have been evaluated by injecting tumorigenic squamous carcinoma cells (SCC-9) overexpressing ADAM17 in immunodeficient mice. RESULTS: The proteomic analysis has effectively identified a total of 2,194 proteins in control and tumor tissues. Among these, 110 proteins have been down-regulated and 90 have been up-regulated in tumor tissues. Biological network analysis has uncovered that overexpression of ADAM17 regulates Erk pathway in OSCC and further indicates proteins regulated by the overexpression of ADAM17 in the respective pathway. These results are also supported by the evidences of higher viability, migration, adhesion and proliferation in SCC-9 or A431 cells in vitro along with the increase of tumor size and proliferative activity and higher tissue collagenase activity as an outcome of ADAM17 overexpression. CONCLUSION: These findings contribute to understand the role of ADAM17 in oral cancer development and as a potential therapeutic target in oral cancer. In addition, our study also provides the basis for the development of novel and refined OSCC-targeting approaches.
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Proteínas ADAM/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Western Blotting , Carcinoma de Células Escamosas/genética , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Bucais/genética , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
BACKGROUND: Although genome-wide association studies have identified several susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in populations around the world, the role of most loci is unknown in the highly heterogeneous Brazilian population. METHODS: To determine the association of 7 markers that showed genome-wide significant association in Brazilians with NSCL/P, we conducted a structured association study conditioned upon the individual ancestry proportions to evaluate markers at 1p36 (rs742071), 2p21 (rs7590268), 3p11.1 (rs7632427), 8q21.3 (rs12543318), 13q31.1 (rs8001641), 15q22.2 (rs1873147), and 17q22 (rs227731) in 505 patients with NSCL/P and 594 healthy controls recruited from 2 different geographical regions of Brazil. The polymorphisms were genotyped by TaqMan 5'-exonuclease allelic discrimination assay, and each sample was independently typed for 40 biallelic short insertion/deletion markers to characterize the genomic ancestry. RESULTS: After Bonferroni correction for multiple tests, significant associations with NSCL/P were observed for rs742071, rs1873147, and rs227731. However, the frequency of the risk alleles varied between the geographical regions, according to the proportions of European and African genomic ancestry. The group enriched by European ancestry showed significant association with rs227731 (p = 0.001), whereas the group with high African ancestry was significantly associated with rs1873147 polymorphism (p = 0.005). The significant association with rs742071 was only detected in the combined sample (p = 0.005). CONCLUSION: The findings of the present study revealed the associations of 1p36 (rs742071), 15q22 (rs1873147), and 17p22 (rs227731) with NSCL/P in the Brazilian population, and further confirmed that the genetic heterogeneity of NSCL/P may be related to the different ethnic background of the affected individuals.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , População Negra , Brasil , Estudos de Casos e Controles , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 17 , Fenda Labial/etnologia , Fenda Labial/patologia , Fissura Palatina/etnologia , Fissura Palatina/patologia , Frequência do Gene , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Padrões de Herança , Mutagênese Insercional , Razão de Chances , População BrancaRESUMO
BACKGROUND: Polymorphisms within the MTHFR (rs2274976) and MTHFD1 (rs2236225) genes were previously associated with maternal susceptibility for having an offspring with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in the Brazilian population. However, as the genotypes of the patients with NSCL/P were not evaluated, it is not clear whether the effects are associated with maternal or offspring genotypes. The aim of this study was to evaluate the association of rs2274976 and rs2236225 in the pathogenesis of NSCL/P. METHODS: By using the TaqMan 5'-exonuclease allelic discrimination assay, the present study genotyped the rs2274976 and rs2236225 polymorphisms in 147 case-parent trios, 181 isolated samples of NSCL/P and 478 healthy controls of the Brazilian population. Transmission disequilibrium test and structured case-control analysis based on the individual ancestry proportions were performed. RESULTS: The transmission disequilibrium test showed a significant overtransmission of the rs2274976 A allele (p = 0.004), but no preferential parent-of-origin transmission was detected. The structured case-control analysis supported those findings, revealing that the minor A allele of rs2274976 was significantly more frequent in NSCL/P group compared with control group (p = 0.001), yielding an odds ratio of 3.46 (95% confidence interval, 2.05-5.85). No association of rs2236225 polymorphism with NSCL/P was observed in both transmission disequilibrium test and case-control analysis. CONCLUSION: The results of the study revealed that the presence of the rs2274976 A allele is a risk marker for the development of NSCL/P in the Brazilian population.
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Fenda Labial/genética , Fissura Palatina/genética , Padrões de Herança , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , Adulto , Alelos , Brasil , Estudos de Casos e Controles , Criança , Fenda Labial/patologia , Fissura Palatina/patologia , Feminino , Frequência do Gene , Ligação Genética , Marcadores Genéticos , Genótipo , Humanos , Masculino , Razão de Chances , RiscoRESUMO
BACKGROUND: The histogenesis of neoplastic spindle cells of Kaposi's sarcoma is still uncertain, but some studies consider it a lymphatic vessel differentiation. Prox-1 is a nuclear transcription factor that plays a major role during embryonic lymphangiogenesis, and it has been considered a specific and sensitive lymphatic endothelial cell marker. The aim of this study was to determine the expression of Prox-1 in oral Kaposi's sarcoma comparing the results with oral benign vascular tumors including capillary hemangiomas and pyogenic granulomas. METHODS: Expression of Prox-1 and HHV-8 was evaluated by immunohistochemistry in 30 oral Kaposi's sarcoma, 5 oral capillary hemangiomas, and 10 oral pyogenic granulomas. The labeling index was expressed as the percentage of positive cells for each case studied. Statistical comparison was performed using the Wilcoxon-Mann-Whitney rank sum test. RESULTS: Twenty-eight (93.3%) and 30 oral Kaposi's sarcoma cases were positive for Prox-1 and HHV-8, respectively, while all oral benign vascular tumors were negative for these markers. The number of Prox-1 and HHV-8 oral Kaposi's sarcoma-positive cells increased significantly from patch/plaque to nodular histological stages. CONCLUSION: The expression of Prox-1 in the neoplastic spindle cells supports the view of a lymphatic differentiation in oral Kaposi's sarcoma. Prox-1 may also be involved in the pathogenesis of oral Kaposi's sarcoma as the number of positive spindle cells increased progressively from patch to nodular stages and could be eventually useful as an additional diagnostic tool for differential diagnosis between oral Kaposi's sarcoma and benign oral vascular lesions.
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Proteínas de Homeodomínio/análise , Neoplasias Bucais/patologia , Sarcoma de Kaposi/patologia , Proteínas Supressoras de Tumor/análise , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Diferenciação Celular , Proliferação de Células , Criança , Endotélio Linfático/patologia , Feminino , Doenças da Gengiva/patologia , Granuloma Piogênico/patologia , Soropositividade para HIV/patologia , Hemangioma Capilar/patologia , Herpesvirus Humano 8/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/virologia , Sarcoma de Kaposi/virologia , Doenças da Língua/patologia , Neoplasias da Língua/patologia , Adulto JovemRESUMO
BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common orofacial birth defect with a wide range prevalence among different populations. Previous association studies with populations from Europe and Asia have identified putative susceptibility markers for NSCL/P in fibroblast growth factor 12 (FGF12), vinculin (VCL), connexin 43 (CX43) and in a region close to the ventral anterior homeobox 1 (VAX1) gene. However, there have thus far been no studies of these markers in NSCL/P Brazilian patients, and as the genetic ancestry of the Brazilian population is highly varied, the predisposition to those disease markers can be different. METHODS: Herein we conducted a structured association study conditioned on the individual ancestry proportions to determine the role of 16 polymorphic markers within those genes in 300 patients with NSCL/P and 385 unaffected controls. RESULTS: None of the alleles and genotypes showed association with NSCL/P, though there was a significant association of the haplotype formed by VAX1 rs10787760, rs6585429 and rs1871345 polymorphisms with NSCL/P that did not persist Bonferroni correction for multiple tests. CONCLUSIONS: Our results are consistent with a lack of involvement of FGF12, VCL and CX43 variants with NSCL/P pathogenesis in Brazilian patients. Furthermore, the higher frequency of a haplotype of VAX1 with NSCL/P patients suggests a low penetrant gene for oral cleft, and warrants further studies.
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Fenda Labial/genética , Fissura Palatina/genética , Conexina 43/genética , Fatores de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Vinculina/genética , Alelos , Brasil , Estudos de Casos e Controles , Fenda Labial/complicações , Fissura Palatina/complicações , Feminino , Variação Genética , Genótipo , Haplótipos , Humanos , Masculino , Razão de Chances , RiscoRESUMO
BACKGROUND AND OBJECTIVE: Nonsyndromic cleft lip and/or palate (NSCL/P) is a complex disease associated with both genetic and environmental factors. One strategy for identifying of possible NSCL/P genetic causes is to evaluate polymorphic variants in genes involved in the craniofacial development. DESIGN: We carried out a case-control analysis of 13 single nucleotide polymorphisms in 9 genes related to craniofacial development, including TBX1, PVRL1, MID1, RUNX2, TP63, TGFß3, MSX1, MYH9 and JAG2, in 367 patients with NSCL/P and 413 unaffected controls from Brazil to determine their association with NSCL/P. RESULTS: Four out of 13 polymorphisms (rs28649236 and rs4819522 of TBX1, rs7940667 of PVRL1 and rs1057744 of JAG2) were presented in our population. Comparisons of allele and genotype frequencies revealed that the G variant allele and the AG/GG genotypes of TBX1 rs28649236 occurred in a frequency significantly higher in controls than in the NSCL/P group (OR: 0.41; 95% CI: 0.25-0.67; p=0.0002). The frequencies of rs4819522, rs7940667 and rs1057744 minor alleles and genotypes were similar between control and NSCL/P group, without significant differences. No significant associations among cleft types and polymorphisms were observed. CONCLUSION: The study suggests for the first time evidences to an association of the G allele of TBX1 rs28649236 polymorphism and NSCL/P.
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Fenda Labial/epidemiologia , Fenda Labial/genética , Fissura Palatina/epidemiologia , Fissura Palatina/genética , Polimorfismo Genético , Brasil/epidemiologia , Estudos de Casos e Controles , Genótipo , Crescimento/genética , Humanos , Desenvolvimento Maxilofacial/genética , Risco , Crânio/crescimento & desenvolvimentoRESUMO
BACKGROUND: HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer. METHODS: In order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression. RESULTS: We showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026). CONCLUSION: Our findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/metabolismo , Neoplasias Bucais/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adesão Celular/fisiologia , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Fatores de Transcrição/fisiologiaRESUMO
Cellular levels of key regulatory proteins are controlled via ubiquitination and subsequent degradation. Deubiquitinating enzymes or isopeptidases can potentially prevent targeted destruction of protein substrates through deubiquitination prior to proteasomal degradation. However, only one deubiquitinating enzyme to date has been matched to a specific substrate in mammalian cells and shown to functionally modify it. Here we show that the isopeptidase USP2a (ubiquitin-specific protease-2a) interacts with and stabilizes fatty acid synthase (FAS), which is often overexpressed in biologically aggressive human tumors. Further, USP2a is androgen-regulated and overexpressed in prostate cancer, and its functional inactivation results in decreased FAS protein and enhanced apoptosis. Thus, the isopeptidase USP2a plays a critical role in prostate cancer cell survival through FAS stabilization and represents a therapeutic target in prostate cancer.
Assuntos
Androgênios/metabolismo , Endopeptidases/metabolismo , Ácido Graxo Sintases/metabolismo , Neoplasias da Próstata/enzimologia , Animais , Cromatografia de Afinidade , Cisteína Endopeptidases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Masculino , Espectrometria de Massas , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , RNA Interferente Pequeno/metabolismo , Ratos , Ubiquitina TiolesteraseRESUMO
OBJECTIVE: Fatty acid synthase levels are associated with aggressiveness, prognosis, and risk of metastasis in oral squamous cell carcinomas. This enzyme contains seven catalytic domains and its inhibition by synthetic or natural drugs has antineoplastic properties such as C75, which is a synthetic inhibitor of the ß- ketoacyl synthase domain, the antibiotic triclosan, ligand of the enoyl reductase domain, and the antiobesity drug orlistat, which inhibits the thioesterase domain. Here, we sought to investigate and compare the in vitro effects of C75, triclosan, and orlistat on malignant phenotypes of the cell line SCC-9: proliferation, cell cycle, apoptosis, adhesion, migration, and invasion. DESIGN: Half-maximal inhibitory concentration (IC50) was determined using cell viability assays. Cell death and cell cycle progression were analyzed by Annexin V-PE/7-ADD-PerCP labeling and propidium iodide staining, respectively. Cell migration and invasion were assayed by transwells assays and cell adhesion using collagen and fibronectin. RESULTS: C75 showed the lowest IC50 and higher inhibition of lipid droplets at low concentrations and reduced cell motility. Triclosan showed the intermediate IC50 value, excellent reduction of lipid bodies at the IC50 when compared with C75 and orlistat. Also, triclosan reduced cell cycle progression, adhesion, migration, and invasion of SCC-9 and induced the highest levels of apoptosis. Orlistat promoted cell cycle arrest, but showed the lowest induction of apoptosis and did not affected invasion and adhesion of SCC-9. CONCLUSION: Altogether, despite the particular effects of the analyzed fatty acid synthase inhibitors, triclosan showed to better interfere in tumorigenic phenotypes of SCC-9 cells.
Assuntos
Ácido Graxo Sintases , Neoplasias Bucais , Apoptose , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/tratamento farmacológico , Orlistate , FenótipoRESUMO
Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Cerulenina/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Inibidores da Síntese de Ácidos Graxos/farmacologia , Lactonas/farmacologia , Melanoma/enzimologia , Análise de Variância , Animais , Apoptose/fisiologia , Cálcio/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Lipídeos/biossíntese , Melanoma/fisiopatologia , Camundongos , Orlistate , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria.
Assuntos
Ácido Graxo Sintases/antagonistas & inibidores , Espectrometria de Massas/métodos , Melanoma/enzimologia , Mitocôndrias/enzimologia , Animais , Linhagem Celular Tumoral , Etanol/química , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Lactonas/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Orlistate , Análise de Componente PrincipalRESUMO
PURPOSE: Fatty acid synthase (FASN), the multifunctional enzyme responsible for endogenous fatty acid synthesis, is highly expressed and associated with poor prognosis in several human cancers, including melanoma. Our group has previously shown that pharmacological inhibition of FASN with orlistat decreases proliferation, promotes apoptosis, and reduces the metastatic spread of B16-F10 cells in experimental models of melanoma. While most of the orlistat antitumor properties seem to be closely related to direct effects on malignant cells, its impact on the host immune system is still unknown. METHODS: The effects of orlistat on the phenotype and activation status of infiltrating leukocytes in primary tumors and metastatic lymph nodes were assessed using a model of spontaneous melanoma metastasis (B16-F10 cells/C57BL/6 mice). Cells from the primary tumors and lymph nodes were mechanically dissociated and immune cells phenotyped by flow cytometry. The expression of IL-12p35, IL-12p40, and inducible nitric oxide synthase (iNOS) was analyzed by qRT-PCR and production of nitrite (NO2-) evaluated in serum samples with the Griess method. RESULTS: Orlistat-treated mice exhibited a 25% reduction in the number of mediastinal lymph node metastases (mean 3.96 ± 0.78, 95% CI 3.63-4.28) compared to the controls (mean 5.7 ± 1.72; 95% CI 5.01-6.43). The drug elicited an antitumor immune response against experimental melanomas by increasing maturation of intratumoral dendritic cells (DC), stimulating the expression of cytotoxicity markers in CD8 T lymphocytes and natural killer (NK) cells, as well as reducing regulatory T cells (Tregs). Moreover, the orlistat-treatment increased serum levels of nitric oxide (NO) concentrations. CONCLUSION: Taken together, these findings suggest that orlistat supports an antitumor response against experimental melanomas by increasing CD80/CD81-positive and IL-12-positive DC populations, granzyme b/NKG2D-positive NK populations, and perforin/granzyme b-positive CD8 T lymphocytes as well as reducing Tregs counts within experimental melanomas.