RESUMEN
Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and ß1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).
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Factor 7 de Crecimiento de Fibroblastos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Diferenciación Celular , Células Cultivadas , Colágeno/metabolismo , Epitelio , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granulocitos/metabolismo , Humanos , Queratinocitos , Macrófagos/metabolismoRESUMEN
Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts' motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFß1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.
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Background: Knowledge on the role of miR changes in tumor stroma for cancer progression is limited. This study aimed to investigate the role of miR dysregulation in cancer-associated fibroblasts (CAFs) in oral squamous cell carcinoma (OSCC). Methodology: CAF and normal oral fibroblasts (NOFs) were isolated from biopsies of OSCC patients and healthy individuals after informed consent and grown in 3D collagen gels. Total RNA was extracted. Global miR expression was profiled using Illumina version 2 panels. The functional impact of altered miR-204 expression in fibroblasts on their phenotype and molecular profile was investigated using mimics and inhibitors of miR-204. Further, the impact of miR-204 expression in fibroblasts on invasion of adjacent OSCC cells was assessed in 3D-organotypic co-cultures. Results: Unsupervised hierarchical clustering for global miR expression resulted in separate clusters for CAF and NOF. SAM analysis identified differential expression of twelve miRs between CAF and NOF. Modulation of miR-204 expression did not affect fibroblast cell proliferation, but resulted in changes in the motility phenotype, expression of various motility-related molecules, and invasion of the adjacent OSCC cells. 3' UTR miR target reporter assay showed ITGA11 to be a direct target of miR-204. Conclusions: This study identifies differentially expressed miRs in stromal fibroblasts of OSCC lesions compared with normal oral mucosa and it reveals that one of the significantly downregulated miRs in CAF, miR-204, has a tumor-suppressive function through inhibition of fibroblast migration by modulating the expression of several different molecules in addition to directly targeting ITGA11.
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Biomarcadores de Tumor/metabolismo , Fibroblastos Asociados al Cáncer/patología , Regulación Neoplásica de la Expresión Génica , Cadenas alfa de Integrinas/metabolismo , MicroARNs/genética , Neoplasias de la Boca/patología , ARN Circular/genética , Apoptosis , Biomarcadores de Tumor/genética , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Humanos , Cadenas alfa de Integrinas/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica , Pronóstico , Células Tumorales CultivadasRESUMEN
BACKGROUND: The objective of this study was to assess patient-reported outcomes such as satisfaction and quality of life after advanced alveolar bone augmentation with anterior iliac crest grafting and implant treatment in orally compromised patients. METHODS: This cross-sectional retrospective cohort study included 59 patients (29 women and 30 men) with major functional problems, who underwent advanced alveolar augmentation with autologous iliac bone grafts during a 100-year period (2002-2012). The self-administered questionnaire included 36 validated questions related to (1) demographics, (2) perceived general and oral health, (3) donor site and hospitalization, (4) status of implants and/or prosthesis, and (5) oral health-related quality of life (OHRQoL). RESULTS: Questionnaires were completed by 44 patients: 24 women and 20 men (response rate, 74.6%). Most patients reported good tolerance of the operative iliac bone harvesting (85%) and implant (90%) procedures. Post-operative pain at the donor site was reported by 38%, lasting 18.1 ± 16.1 days. An average of 4.3 ± 3.5 days of hospitalization and 20.2 ± 18.5 days of sick leave was reported. The overall satisfaction with prosthetic reconstruction was 90.5%. OHRQoL was reported with a mean Oral Health Impact Profile-14 (OHIP-14) score of 8.4. CONCLUSION: Favorable OHRQoL and satisfaction were reported after advanced reconstruction of alveolar ridges with iliac crest-derived grafting and implants in severely compromised patients. However, this treatment requires substantial resources including hospitalization and sick leave.
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The epithelial rests of Malassez (ERMs) might represent a valuable source of oral epithelial cells with stem cell properties. The purpose of this study was to isolate and characterize cells derived from human ERM, and compare them with cells derived from matched normal oral mucosa (NOM). Matched tissue specimens of the periodontal ligament of extracted tooth and NOM were collected. Cells were isolated in culture, then characterized by immunohistochemistry and flow cytometry for expression of pancytokeratin, ESA, PDGFRB, CD31 and CD44. 3D organotypic cultures were constructed by growing epithelial cells on top of fibroblast-populated collagen gels. Both ERM and NOM-isolated cells expressed the markers of epithelial lineage (ESA and pancytokeratin), and to some extent PDGFR, an indicator of a more mesenchymal phenotype, but not the endothelial cell marker CD31. Cells with epithelial morphology were isolated from periodontium of cervical, middle and apical parts of the root, but contained a significantly lower percentage of ESA and pancytokeratin-positive cells than when isolating cells from NOM (p < 0.001). ERM cells expressed a significantly higher percentage of the stem cell-related molecule CD44 (cervical 92.93 ± 0.25%, middle 93.8 ± 0.26%, apical 94.36 ± 0.41%) than cells isolated from NOM (27.8 ± 1.47%, p < 0.001). When grown in 3D organotypic cultures and in collagen gels, ERM cells formed a less differentiated epithelium than NOM cells, but expressing pancytokeratin and vimentin. In conclusion, epithelial cells could be isolated from human periodontium and grown in culture; their in vitro characterization indicates that they have a less differentiated phenotype compared with cells derived from normal oral epithelium.
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Ligamento Periodontal , Descanso , Células Cultivadas , Células Epiteliales , Fibroblastos , HumanosRESUMEN
BACKGROUND: Oral cavity is a doorway for a variety of products containing titanium dioxide (TiO2 ) nanoparticles (NPs) (nano-TiO2 ) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined. OBJECTIVES: To evaluate the ability of nano-TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM). METHODS: RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano-TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh-resolution imaging (URI) microscopy (CytoViva™ , Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues. RESULTS: Ultrahigh-resolution imaging microscopy demonstrated the presence of nano-TiO2 mostly in the epithelium of RNHBM at both 20 min and 24-h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness. CONCLUSION: Nano-TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze- and time-dependent manner.
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Mucosa Bucal/metabolismo , Titanio/farmacocinética , Humanos , Técnicas In Vitro , Nanopartículas del Metal , Microscopía , Tamaño de la Partícula , PermeabilidadRESUMEN
BACKGROUND: Cancer-associated fibroblasts (CAFs) were shown to be important for tumour progression in head and neck squamous cell carcinomas (HNSCCs). Their heterogeneity and lack of specific markers is increasingly recognized. Integrin α11 was recently shown to be expressed by CAFs and might serve as a specific CAF marker. AIM: To investigate integrin α11 expression and its correlation with the expression of a well-known marker of CAF, alpha smooth muscle actin (α-SMA), in HNSCC. METHODS: Fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples from healthy volunteers (n = 24), oral lichen planus (OLP) (n = 32) and HNSCC (n = 106) were collected together with clinical data after ethical approval. Immunohistochemistry to detect integrin α11 and α-SMA was performed on FF and FFPE samples. qPCR for integrin α11 (ITGA11) and α-SMA (ACTA2) was performed on FF samples. Data were analysed using chi-square test and Kaplan-Meier survival analysis. RESULTS: Significantly higher levels of integrin α11 and α-SMA at both protein and mRNA levels were found in HNSCC vs. normal controls and OLP. A strong correlation was found between integrin α11 and α-SMA expression, and double staining showed their colocalization. Both integrin α11 and α-SMA were detected surrounding metastatic islands. Expression of α-SMA at tumour front but not tumour centre correlated with patient survival. CONCLUSION: Integrin α11 was overexpressed in HNSCC stroma and colocalized with α-SMA. Expression of α-SMA at tumour front but not tumour centre had prognostic value for survival, pinpointing the importance of assessing tumour front when evaluating stromal molecules as prognostic biomarkers.
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Actinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Cadenas alfa de Integrinas/metabolismo , Músculo Liso/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: Although several approaches for identification and isolation of carcinoma cells with tumour initiating properties have been established, enrichment of a population of pure and viable tumour-initiating cells (TICs) is still problematic. This study investigated possibilities to isolate a population of cancer cells with tumour initiating properties based on their adherence properties, rather than expression of defined markers or clonogenicity. METHODS: Several human cell lines derived from oral dysplasia and oral squamous cell carcinoma (OSCC), as well as primary cells derived from patients with OSCC were allowed to adhere to collagen IV-coated dishes sequentially. Rapid adherent cells (RAC), middle adherent cells (MAC) and late adherent cells (LAC) were then harvested and further investigated for their morphology, stem cell-like properties and molecular profile while grown in vitro and tongue xenotransplantation in NOD-SCID mice at serial dilutions. RESULTS: RAC showed significantly higher colony forming efficiency (p<0.05), sphere forming ability, greater migration ability (p<0.05), exhibited longer G2 phase and displayed higher expression of integrin ß1 and other stem-cell related molecules as compared to MAC and LAC. RAC induced tongue tumours in NOD-SCID mice with the highest incidence. These tumours were also bigger and metastasised more frequently in loco-regional lymph nodes than MAC and LAC. CONCLUSIONS: These findings prove for the first time that OSCC cells with tumour initiating properties can be enriched based on their rapid adhesiveness to collagen IV. This separation procedure provides a potentially useful tool for isolating TICs in OSCC for further studies on understanding their characteristics and drug-resistant behaviour.
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Carcinoma de Células Escamosas/fisiopatología , Colágeno Tipo IV/farmacología , Neoplasias de la Boca/fisiopatología , Células Madre Neoplásicas/fisiología , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis/patología , Carcinoma de Células Escamosas/patología , Adhesión Celular/fisiología , Línea Celular Transformada , Humanos , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Boca/patología , Trasplante de Neoplasias/métodos , Células Madre Neoplásicas/patología , Trasplante Heterólogo/métodos , Regulación hacia ArribaRESUMEN
BACKGROUND: Epigenetic reprogramming of the methylome has been implicated in all stages of cancer evolution. It is now well accepted that cancer cells exploit epigenetic reprogramming, a mechanism that regulates stem/progenitor cell renewal and differentiation, to promote cancer initiation and progression. The oncogene FOXM1 has been implicated in all major forms of human cancer. METHODS: We have recently shown that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a number of FOXM1-induced epigenetic markers. Differential promoter methylation and gene expression in clinical specimens were measured using commercially available bisulfite conversion kits and absolute quantitative PCR, respectively. RESULTS: Here, we investigated 8 FOXM1-induced differentially methylated genes, SPCS1, FLNA, CHPF, GLT8D1, C6orf136, MGAT1, NDUFA10, and PAFAH1B3, using human head and neck tissue specimens donated by 2 geographically independent patient cohorts from Norway and the United Kingdom. Two genes (GLT8D1 and C6orf136) were found to be differentially expressed in head and neck squamous cell carcinomas (HNSCCs). Using methylation-specific quantitative PCR, we confirmed that the promoters of GLT8D1 and C6orf136 were hypo- and hypermethylated, respectively, in HNSCC tissues. CONCLUSIONS: Given that epigenetic change precedes gene expression, methylation status of candidate genes identified from this study may represent a signature of premalignancy, rendering them potentially useful predictive biomarkers for precancer screening and/or therapeutic targets for cancer prevention.
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Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Factores de Transcripción Forkhead/genética , Neoplasias de Cabeza y Cuello/genética , Metilación de ADN , Epigénesis Genética , Femenino , Proteína Forkhead Box M1 , Regulación Neoplásica de la Expresión Génica , Glicosiltransferasas/genética , Humanos , Masculino , Persona de Mediana Edad , Noruega , Regiones Promotoras Genéticas , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello , Reino Unido , Regulación hacia ArribaRESUMEN
Histopathological discordance with molecular phenotype of many human cancers poses clinically challenging tasks for accurate cancer diagnosis, which impacts on treatment strategy and patient outcome. Hence, an objective, accurate and quantitative method is needed. A quantitative Malignancy Index Diagnostic System (qMIDS) was developed based on 14 FOXM1 (isoform B)-associated genes implicated in the regulation of the cell cycle, differentiation, ageing, genomic stability, epigenetic and stem cell renewal, and two reference genes. Their mRNA expression levels were translated via a prospectively designed algorithm, into a metric scoring system. Subjects from UK and Norway (n = 299) provided 359 head and neck tissue specimens. Diagnostic test performance was assessed using detection rate (DR) and false-positive rate (FPR). The median qMIDS scores were 1.3, 2.9 and 6.7 in healthy tissue, dysplasia and head and neck squamous cell carcinomas (HNSCC), respectively (UK prospective dataset, p<0.001); 1.4, 2.3 and 7.6 in unaffected, oral lichen planus, or HNSCC, respectively (Norwegian retrospective dataset with up to 19 years survival data, p<0.001). At a qMIDS cut-off of 4.0, DR was 94% and FPR was 3.2% (Norwegian dataset); and DR was 91% and FPR was 1.3% (UK dataset). We further demonstrated the transferability of qMIDS for diagnosing premalignant human vulva (n = 58) and skin (n = 21) SCCs, illustrating its potential clinical use for other cancer types. This study provided evidence that qMIDS was able to quantitatively diagnose and objectively stratify cancer aggressiveness. With further validation, qMIDS could enable early HNSCC detection and guide appropriate treatment. Early treatment intervention can lead to long-term reduction in healthcare costs and improve patient outcome.
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Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/diagnóstico , Factores de Transcripción Forkhead/genética , Neoplasias de Cabeza y Cuello/diagnóstico , Lesiones Precancerosas/diagnóstico , Neoplasias Cutáneas/diagnóstico , Neoplasias de la Vulva/diagnóstico , Algoritmos , Carcinoma de Células Escamosas/genética , Células Cultivadas , Diagnóstico Precoz , Femenino , Proteína Forkhead Box M1 , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Neoplasias de Cabeza y Cuello/genética , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Metástasis Linfática , Masculino , Persona de Mediana Edad , Noruega , Lesiones Precancerosas/genética , Pronóstico , Estudios Prospectivos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/genética , Neoplasias de la Vulva/genéticaRESUMEN
OBJECTIVE: Fusobacterium nucleatum is an opportunistic pathogen with a key role in subgingival plaque formation and it is found in increased numbers in periodontally affected sites. This study aimed to investigate the potential of F. nucleatum to penetrate and induce alterations in an in vitro reconstructed human gingival mucosa model. METHODS: Three-dimensional (3D) organotypic models of human gingiva were engineered using primary gingival keratinocytes and fibroblasts. The reconstructed tissues were challenged with four different strains of fluorescently labelled F. nucleatum in suspension placed on top of epithelial layers. Confocal laser scanning was used to assess the presence of fusobacteria through the organotypic model. Apoptosis (cleaved caspase-3) and cell proliferation (Ki-67) were evaluated by the use of immunohistochemistry in 3D-tissue models. Quantitative real-time PCR was performed to investigate the mRNA expression for MMP-13 and E-cadherin in both 3D-tissues and monolayers. RESULTS: F. nucleatum invaded the superficial epithelial layers of gingival 3D-tissue models. Challenged tissues showed accentuated shedding of superficial layers and increased number of cleaved caspase-3 and Ki-67 positive cells than controls, although not statistically significant. Levels of E-cadherin and MMP-13 mRNA were not significantly perturbed in multilayer culture. A variable and disproportionate response of MMP-13 mRNA level resulted in challenged primary keratinocytes in monolayers, compared to multilayer culture. CONCLUSION: These results indicate that F. nucleatum is able to invade superficially a differentiated, stratified gingival epithelium in vitro and triggers the efficient elimination of bacterial infection through epithelial shredding without causing a permanent damage of the tissue.
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Apoptosis/fisiología , Cadherinas/metabolismo , Caspasa 3/metabolismo , Células Epiteliales/microbiología , Fusobacterium nucleatum/patogenicidad , Encía/microbiología , Enfermedades Periodontales/metabolismo , Análisis de Varianza , Células Cultivadas , Células Epiteliales/metabolismo , Fusobacterium nucleatum/inmunología , Encía/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Microscopía Confocal , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
PURPOSE: The aim of this prospective study was to evaluate the long-term clinical and radiographic outcomes of nonsurgically treated mandibular condylar fractures in children. MATERIALS AND METHODS: This study includes 42 children (23 girls and 19 boys) with 54 mandibular condylar fractures. All patients were younger than 18 years at the time of injury and were followed for at least 2 years (mean follow-up 4 years). Treatment was either observation or intermaxillary fixation (rigid and/or guiding elastics). Clinical outcome was categorized as favorable or unfavorable. Condylar remodeling was defined as complete, moderate, or poor on the basis of radiographic findings. RESULTS: Thirty-one patients (74%) presented with favorable and 11 (26%) with unfavorable clinical long-term outcome. Unilateral fractures showed a statistically significant increase of unfavorable clinical outcomes compared with bilateral fractures (P = .018). The radiologic examinations showed complete remodeling in 47 (87%), moderate remodeling in 5 (9%), and poor remodeling in 2 (4%) of the condylar fractures. One patient developed condylar overgrowth. No statistically significant relationship between clinical and radiologic outcome was found. Thirty-five patients (83%) had no subjective symptoms, and 41 (98%) described normal chewing function at the last follow-up examination. CONCLUSIONS: Nonsurgical treatment of mandibular condylar fractures leads to acceptable remodeling of the condylar process, good clinical long-term outcome, and minimal subjective symptoms in most children. Unilateral fractures significantly increase the risk for unfavorable clinical outcome.
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Cóndilo Mandibular/lesiones , Fracturas Mandibulares/terapia , Adolescente , Remodelación Ósea/fisiología , Niño , Preescolar , Oclusión Dental , Dolor Facial/clasificación , Femenino , Estudios de Seguimiento , Curación de Fractura/fisiología , Humanos , Técnicas de Fijación de Maxilares/instrumentación , Luxaciones Articulares/clasificación , Luxaciones Articulares/diagnóstico por imagen , Luxaciones Articulares/terapia , Estudios Longitudinales , Masculino , Cóndilo Mandibular/diagnóstico por imagen , Fracturas Mandibulares/clasificación , Fracturas Mandibulares/diagnóstico por imagen , Masticación/fisiología , Estudios Prospectivos , Radiografía Panorámica , Rango del Movimiento Articular/fisiología , Factores Sexuales , Resultado del TratamientoRESUMEN
BACKGROUND: The high incidence of oral cancer in Sudan has been associated with the use of toombak, the local type of smokeless tobacco. However, its specific effects on human oral cells are not known. We aimed to investigate the effects of toombak on primary normal human oral keratinocytes, fibroblasts, and a dysplastic oral keratinocytic cell line, and to compare them with the effects induced by Swedish snuff. METHOD: Aqueous extracts were prepared from moist toombak and Swedish snuff and added in serial dilutions on in vitro monolayer cultured cells. Cell viability, morphology and growth, DNA double-strand breaks (gammaH2AX staining), expression of phosphatidylserine (Annexin V staining), and cell cycle were assessed after various exposure time periods. RESULTS: Significant decrease in cell number, occurrence of DNA double-strain breaks, morphological and biochemical signs of programmed cell death were detected in all oral cell types exposed to clinically relevant dilutions of toombak extract, although to a lesser extent in normal oral fibroblasts and dysplastic keratinocytes. G2/M-block was also detected in normal oral keratinocytes and fibroblasts exposed to clinically relevant dilutions of toombak extract. Swedish snuff extract had less adverse effects on oral cells, mainly at non-clinically relevant dilutions. CONCLUSION: This study indicates a potential for toombak, higher than for Swedish snuff, to damage human oral epithelium. Dysplastic oral keratinocytes were less sensitive than their normal counterparts, suggesting that they might have acquired a partially resistant phenotype to toombak-induced cytotoxic effects while still being prone to DNA damage that could lead to further malignant progression.
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Mucosa Bucal/citología , Tabaco sin Humo/efectos adversos , Adulto , Anexina A5/análisis , Apoptosis/efectos de los fármacos , Recuento de Células , División Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Roturas del ADN de Doble Cadena , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fase G2/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Microscopía Electrónica de Transmisión , Mucosa Bucal/efectos de los fármacos , Fosfatidilserinas/análisis , Extractos Vegetales/efectos adversos , Sudán , SueciaRESUMEN
BACKGROUND: Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front. OBJECTIVE: This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial-mesenchymal interactions play on it, by using an in vitro oral cancer progression model. MATERIAL AND METHODS: In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin. RESULTS: Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium-biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin. CONCLUSIONS: These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial-mesenchymal environment, respectively the existence of fibroblasts.
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Membrana Basal/química , Carcinoma de Células Escamosas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Modelos Biológicos , Neoplasias de la Boca/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Comunicación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Colágeno Tipo IV/biosíntesis , Progresión de la Enfermedad , Fibroblastos/fisiología , Fibronectinas/biosíntesis , Humanos , Imagenología Tridimensional , Técnicas para Inmunoenzimas , Queratinocitos/fisiología , Mesodermo/citología , KalininaRESUMEN
Previous studies suggest the use of khat, a psychostimulant plant used by millions of people in Middle East and Africa, as risk factor for oral cancer. We previously reported that khat is able to induce adverse affects, as cell cycle arrest and apoptosis, in normal human oral cells cultured in vitro. This study further investigates the more specific role played by mitochondria in khat-induced cell death and the kinetics of the events involved in this process. Exposure of primary normal human oral keratinocytes and fibroblasts to khat extract resulted in a swift and sustained decrease of the mitochondrial inner transmembrane potential occurring within 0.5-1h. Loss of mitochondrial membrane potential preceded all other biochemical and morphologic changes, and was associated with a significant decrease in cell survival. Subsequently, apoptosis-inducing factor was released from mitochondria into cytosol and relocated to nucleus. Cyclosporine A and bongkrekic acid delayed both the loss of mitochondrial inner transmembrane potential and the onset of cell death. This study describes a novel mechanism of khat-induced cell death in primary normal oral keratinocytes and fibroblasts involving an early pivotal effect on mitochondrial function and integrity.
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Catha/toxicidad , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mucosa Bucal/citología , Adulto , Factor Inductor de la Apoptosis/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Humanos , CinéticaRESUMEN
BACKGROUND: Fusobacterium nucleatum, a commensal opportunistic oral bacterium, is capable of invading gingival epithelial cells, but the entrance into human primary oral fibroblast cells has not been documented. This study evaluated the ability of three strains of F. nucleatum (F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, and F. nucleatum ssp. vincentii) to enter gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PLFs). METHODS: GFs and PLFs were cocultured for various periods of time with different strains of F. nucleatum. Scanning and transmission electron microscopy, together with confocal laser scanning microscopy, were used to visualize the entrance and presence of bacteria in host cells. Flow cytometry was performed to compare the load of internalized bacteria in GFs and PLFs exposed for 3 and 5 hours to live F. nucleatum labeled with fluorescein isothiocyanate. RESULTS: All three strains of F. nucleatum were found entering and located in the cytoplasm of GFs and PLFs after 1 hour of exposure. Flow cytometry tests revealed a significant increase in the fluorescent signal, compared to baseline, derived from bacteria internalized in fibroblasts exposed for 3 hours (P <0.001); a further increase was found at 5 hours. The greatest bacterial mass in exposed fibroblasts of both types was of F. nucleatum ssp. polymorphum; the smallest was of F. nucleatum ssp. vincentii. Although not statistically significant, PLFs had a higher bacterial load than corresponding GFs. CONCLUSION: F. nucleatum was capable of entering GFs and PLFs in a manner that is dependent on the cell type and the bacterial strain.
Asunto(s)
Fibroblastos/microbiología , Fusobacterium nucleatum/fisiología , Interacciones Huésped-Patógeno/fisiología , Adhesión Bacteriana/fisiología , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/fisiología , Infecciones por Fusobacteriaceae/microbiología , Fusobacterium nucleatum/patogenicidad , Encía/citología , Humanos , Valores de Referencia , Especificidad de la Especie , VirulenciaRESUMEN
Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3-6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Catha/toxicidad , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Mucosa Bucal/efectos de los fármacos , Extractos Vegetales/toxicidad , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Microscopía Electrónica de Transmisión , Mucosa Bucal/citología , Especies Reactivas de OxígenoRESUMEN
Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16(INK4A) (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21(WAF1/CIP1) (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo, especially in keratinocytes.
Asunto(s)
Catha/efectos adversos , Fibroblastos/efectos de los fármacos , Genes p16/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Mucosa Bucal/efectos de los fármacos , Extractos Vegetales/efectos adversos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Adulto , Animales , Bovinos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/citología , Fase G1/efectos de los fármacos , Humanos , Queratinocitos/citología , Masculino , Mucosa Bucal/citología , Factores de Tiempo , Proteína p53 Supresora de Tumor/análisisRESUMEN
Sodium lauryl sulphate (SLS) is a common detergent known to cause irritation and inflammatory reactions in skin. SLS is also the most commonly used toothpaste detergent and has been related to intraoral adverse effects. However, its specific biological effects on the oral mucosa (OM) have not yet been identified. The objective of this study was to investigate the putative effects of SLS on human oral epithelium using a novel in vitro reconstructed three-dimensional cell culture model. Reconstructed human OM, generated from primary normal human oral keratinocytes and fibroblasts, was exposed to clinically relevant concentrations of SLS (range 0.015-1.5%). The cultured tissues were evaluated by histomorphometry, immunohistochemistry (Ki-67, epithelial (E)-cadherin, alpha6-, beta1-integrins, cleaved caspase-3) and the TUNEL method. Increased epithelial thickness, enhanced proliferation (Ki-67), a more pronounced expression of E-cadherin throughout all epithelial cell layers and single TUNEL-positive cells in the middle spinous cell layers were observed in cultures exposed to low concentrations (0.015%) of SLS. At exposure to higher SLS concentrations (>or=0.15%), epithelial thickness, cell proliferation and E-cadherin expression gradually decreased and in the central areas of exposed regions, cells detached from each other and underwent cell death. In conclusion, clinically relevant concentrations of SLS have dual effects on reconstituted human OM; although occasional cell death within the epithelium was also observed, the increased epithelial thickness, proliferation and E-cadherin expression induced at lower concentrations might be associated with a protective mucosal response, whereas at higher concentrations a more destructive type of reaction predominated.
Asunto(s)
Cadherinas/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mucosa Bucal/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , Análisis de Varianza , Cadherinas/metabolismo , Muerte Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patología , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/efectos de los fármacos , Antígeno Ki-67/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Dodecil Sulfato de Sodio/administración & dosificación , Tensoactivos/administración & dosificaciónRESUMEN
Oral lichen planus (OLP) may be associated with a small risk of malignant transformation of the oral mucosa. Using cases which had transformed, and those which had not, this study aimed to evaluate the potential of DNA content, expression of Cyclooxygenase-2 (Cox-2) and of epithelial (E)-cadherin as risk markers in lesions of OLP. We investigated 78 archival biopsies from; (1) 26 OLP patients with at least two biopsies, of whom seven presented OLP with epithelial dysplasia, followed by oral squamous cell carcinoma (OSCC) in five of them, (2) 19 OLP patients with one biopsy taken. Image cytometry for measurement of DNA content and immunohistochemistry for visualisation of Cox-2 and E-cadherin expression were performed. All OLP biopsies investigated were classified as diploid, one OLP with epithelial dysplasia was tetraploid and all OSCC were diploid. Cox-2 was detected in the epithelium of all OLP specimens investigated, as well as in epithelial dysplasias and OSCC. Focal loss of E-cadherin expression was observed in basal keratinocytes in 88% of the OLP specimens investigated, in all epithelial dysplasias and OSCC. In conclusion, neither aneuploidy, Cox-2 expression, nor loss of E-cadherin expression, were significant reliable markers to select OLP lesions at risk for development of OSCC in the present patient material.