Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of activin A

SummaryMyofibroblasts are essential during wound healing and are often found in the stroma of oral squamous cell carcinomas (OSCC). Although the molecular mechanisms by which myofibroblasts influence OSCC remain largely unknown, previous studies demonstrated that presence of myofibroblast in OSCC stroma is an important risk factor of patient’s shortened survival. Here we showed that some growth factors are produced in higher levels by tumor-associated myofibroblasts compared to tumor-associated fibroblasts, including activin A. Myofibroblast-conditioned media containing activin A significantly increased OSCC cell proliferation and tumor volume, whereas down-regulation of activin A in the conditioned media decreased proliferation. In addition, myofibroblasts induced in vitro invasion of OSCC cells, which was accompanied by an increased production of matrix metalloproteinases (MMP). In vivo, a significant correlation between presence of myofibroblasts and activities of MMP-2 and MMP-9 was observed in OSCC samples. However, blockage of activin A synthesis by myofibroblasts did not affect invasion and MMP production by OSCC cells. Together, our data demonstrate that activin A is required for the proliferative effects of myofibroblasts on OSCC cells. We conclude that myofibroblasts in the stroma of OSCC may influence proliferation and invasion, resulting in more aggressive tumor

Comparação microscópica e proliferativa de fibroblastos gengivais de pacientes com gengiva normal e com fibromatose gengival hereditária

Hereditary gingival fibromatosis (HGF) is a rare oral condition, clinically manifested through a generalized and fibrotic enlargement of the gingiva, which may present as an isolated clinical finding or in association with other features, as part of a syndrome. The biological mechanisms involved in HGF are unknown, and the results of cell-culture studies are controversial. To elucidate the phenotypic and proliferative characteristics of HGF fibroblasts, we isolated 4 cell lines of gingival fibroblasts from members of the same family with HGF, and compared with gingival fibroblasts from 4 healthy patients (NG). HGF and NG fibroblasts, in subconfluent culture densities, showed typical morphological characteristics, such as spindle form with a central spherical nucleus and long cytoplasmatic prolongations, but in saturation density, HGF cells were shorter than control cells. The nucleus/cytoplasm relation was always smaller in all HGF cell lines, suggesting that the cellular reduction is derived from reduction or compaction of the cytoplasm and not of the nucleus. The proliferation rate was higher in fibroblasts from HGF than in the ones from NG. These results suggest that differences in the morphology and proliferation of HGF fibroblasts may be associated with the biological events involved in the pathogenesis of gingival overgrowth in HGF patients.Fibromatose gengival hereditária (FGH) é uma condição bucal rara clinicamente manifestada por um aumento gengival generalizado e fibrótico, podendo apresentar-se de forma isolada ou associada a outras alterações, como parte de síndromes. Os mecanismos biológicos envolvidos na FGH são desconhecidos, e os resultados de estudos de cultura celulares são controversos. Para elucidar as características fenotípicas dos fibroblastos de FGH, nós isolamos quatro linhagens celulares de fibroblastos de FGH de indivíduos de uma mesma família e comparamos as características morfológicas e proliferativas com fibroblastos provenientes de pacientes com gengiva clinicamente normal (GN). Fibroblastos de GN e FGH em condições de subconfluência celular apresentaram típicas características morfológicas, como formato fusiforme, núcleo central e longos prolongamentos citoplasmáticos, mas em condições de saturação da densidade celular, os fibroblastos de FGH apresentaram dimensões menores que as células controle. A relação núcleo/citoplasma foi sempre menor para todas as linhagens celulares de fibroblastos de FGH, sugerindo que a redução celular, é proveniente de uma redução ou compactação citoplasmática e não nuclear. A capacidade proliferativa de fibroblastos de FGH foi maior que a de fibroblastos de GN. Estes resultados sugerem que diferenças morfológicas e proliferativas dos fibroblastos de FGH podem estar associadas aos eventos biológicos envolvidos na etiopatogenia do aumento gengival observado em pacientes com FGH

Hoxa10 controls proliferation, migration and invasion in oral squamous cell carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10- mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previou8436133623FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)sem informaçãosem informaçãoAlthough HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previo

Goldenhar syndrome: clinical features with orofacial emphasis

OBJECTIVES: Goldenhar syndrome (GS) is a relatively common developmental disorder characterized by craniofacial anomalies in association with vertebral, cardiac, renal, and central nervous system defects. This paper describes GS features with special emphasis on oral characteristics. MATERIAL AND METHODS: The clinical features of 6 patients with GS aged 3 months to 12 years are described, and a brief review of the literature about this genetic disorder is presented. RESULTS: All patients demonstrated the classical triad of GS, including mandibular hypoplasia resulting in facial asymmetry, ear and/or eye malformation, and vertebral anomalies. In addition, renal and gastrointestinal abnormalities were observed in 2 patients. Regarding the oral involvement, 2 patients presented cleft lip and palate, and 1 patient had temporomandibular joint malformation. Malocclusion was found in all patients. CONCLUSION: Our orofacial findings correlate with the reported cases in the literature, and point out that after diagnosis GS patients need to be examined for systemic abnormalities

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas

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 (TM) 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.Peer reviewe

Pharmacological fatty acid synthase inhibitors differently affect the malignant phenotype of oral cancer cells.

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 beta-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.& nbsp;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.& nbsp;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.& nbsp;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.Peer reviewe

Comparison between gingival and periodontal ligament fibroblasts from the same subject

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe objective of this study was to compare fibroblasts from the periodontal ligament (PLF) and gingival fibroblasts (GF) as to morphology, proliferation rate and protein synthesis. PLF and GF were explanted from tissues of the same patient. To characterize and compare the morphology of cells, PLF and GF were plated and analyzed under phase-contrast and optical microscopies. Proliferation rates were determined by means of automated counts carried out in days 1, 4, 7, 15 and 21, and also by means of the bromodeoxyuridine labelling index (BrdU). Total protein content was analyzed by means of electrophoresis in 10% polyacrylamide gel and zimography containing gelatin as substrate. PLF were bigger and more elongated than GF in subconfluence and confluence conditions. The proliferative rate of PLF was higher than that of GF at 1, 4, and 7 days (p < 0.05). At 15 and 21 days, there was no statistically significant difference as to the number of cells. PLF presented a significantly greater proliferative potential, in relation to GF (p < 0.05). The synthesis of protein in a period of 24 hours was similar for both PLF and GF. Our results demonstrated that PLF and GF are different as to morphology and proliferative capacity, however, they do not differ as to protein synthesis.O objetivo deste estudo foi comparar as características morfológicas, o potencial proliferativo e a produção protéica de fibroblastos do ligamento periodontal (FLP) e de fibroblastos gengivais (FG). Os fibroblastos foram cultivados pela técnica do explante a partir de fragmentos da gengiva e do ligamento periodontal de um mesmo indivíduo. As células foram isoladas e plaqueadas para análise por microscopia de contraste de fase e microscopia óptica. O índice de proliferação celular foi determinado por contagem automática de células e pelo ensaio de incorporação de bromodioxiuridina (BrdU). A produção de proteína total foi verificada por eletroforese em gel de poliacrilamida e o perfil enzimático por análise zimográfica. Os FLP são maiores e mais alongados que os FG em condições de subconfluência e confluência celular. Os FLP demonstraram um potencial proliferativo significantemente maior que os FG. Os perfis protéico e enzimático foram similares entre os FLP e FG. Os resultados demonstram que os FLP e FG são diferentes na morfologia e na capacidade proliferativa, porém são semelhantes na produção protéica164319325FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO1999/10767-8; 1999/04921-4The objective of this study was to compare fibroblasts from the periodontal ligament (PLF) and gingival fibroblasts (GF) as to morphology, proliferation rate and protein synthesis. PLF and GF were explanted from tissues of the same patient. To characterize and compare the morphology of cells, PLF and GF were plated and analyzed under phase-contrast and optical microscopies. Proliferation rates were determined by means of automated counts carried out in days 1, 4, 7, 15 and 21, and also by means of the bromodeoxyuridine labelling index (BrdU). Total protein content was analyzed by means of electrophoresis in 10% polyacrylamide gel and zimography containing gelatin as substrate. PLF were bigger and more elongated than GF in subconfluence and confluence conditions. The proliferative rate of PLF was higher than that of GF at 1, 4, and 7 days (p < 0.05). At 15 and 21 days, there was no statistically significant difference as to the number of cells. PLF presented a significantly greater proliferative potential, in relation to GF (p < 0.05). The synthesis of protein in a period of 24 hours was similar for both PLF and GF. Our results demonstrated that PLF and GF are different as to morphology and proliferative capacity, however, they do not differ as to protein synthesi

Comparação entre fibroblastos gengivais e do ligamento periodontal de um mesmo indivíduo

The objective of this study was to compare fibroblasts from the periodontal ligament (PLF) and gingival fibroblasts (GF) as to morphology, proliferation rate and protein synthesis. PLF and GF were explanted from tissues of the same patient. To characterize and compare the morphology of cells, PLF and GF were plated and analyzed under phase-contrast and optical microscopies. Proliferation rates were determined by means of automated counts carried out in days 1, 4, 7, 15 and 21, and also by means of the bromodeoxyuridine labelling index (BrdU). Total protein content was analyzed by means of electrophoresis in 10% polyacrylamide gel and zimography containing gelatin as substrate. PLF were bigger and more elongated than GF in subconfluence and confluence conditions. The proliferative rate of PLF was higher than that of GF at 1, 4, and 7 days (p < 0.05). At 15 and 21 days, there was no statistically significant difference as to the number of cells. PLF presented a significantly greater proliferative potential, in relation to GF (p < 0.05). The synthesis of protein in a period of 24 hours was similar for both PLF and GF. Our results demonstrated that PLF and GF are different as to morphology and proliferative capacity, however, they do not differ as to protein synthesis.O objetivo deste estudo foi comparar as características morfológicas, o potencial proliferativo e a produção protéica de fibroblastos do ligamento periodontal (FLP) e de fibroblastos gengivais (FG). Os fibroblastos foram cultivados pela técnica do explante a partir de fragmentos da gengiva e do ligamento periodontal de um mesmo indivíduo. As células foram isoladas e plaqueadas para análise por microscopia de contraste de fase e microscopia óptica. O índice de proliferação celular foi determinado por contagem automática de células e pelo ensaio de incorporação de bromodioxiuridina (BrdU). A produção de proteína total foi verificada por eletroforese em gel de poliacrilamida e o perfil enzimático por análise zimográfica. Os FLP são maiores e mais alongados que os FG em condições de subconfluência e confluência celular. Os FLP demonstraram um potencial proliferativo significantemente maior que os FG. Os perfis protéico e enzimático foram similares entre os FLP e FG. Os resultados demonstram que os FLP e FG são diferentes na morfologia e na capacidade proliferativa, porém são semelhantes na produção protéica

Contribution of polymorphisms in genes associated with craniofacial development to the risk of nonsyndromic cleft lip and/or palate in the Brazilian population

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, TGFB3, 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