Estudo da ação de nanopartículas magnéticas de ferro no câncer oral

Pós-graduação em Genética - IBILCE

O efeito da melatonina na angiogênese e metástase de linhagens de carcinoma epidermóide bucal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlation of clinical, cytological and histological findings in oral squamous cell carcinomas

The present study aimed to investigate the efficiency of exfoliative cytology by correlating the clinical lesions of oral squamous cell carcinoma (OSCC) with exfoliative cytology and histopathological findings. Cases of OSCC diagnosed between 1984 and 2010 were analyzed. The inclusion criteria for the present study were the availability of detailed clinical findings and a diagnosis of the disease through exfoliative cytology and histopathology. The cases were assessed and assigned scores, which were then submitted to modal expression analysis, which considers the higher frequency scores, thus relating the variables. The cytological findings demonstrated that the majority of the cases had malignant potential. Exfoliative cytology should be used as a supplementary tool for the diagnosis of OSCC, as it enables the early detection of these lesions. However, cytology should not be used as a substitute for histopathological examination.

Nanotechnology-based drug delivery systems for treatment of oral cancer: a review

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão imuno-histoquímica do cd8, foxp3, Tgf β, tnf α e nf-кb em displasias epiteliais e Carcinomas epidermóides orais

The Oral Epithelial Dysplasia (OED) is the lesion that precedes or co-exists with the Oral Squamous Cell Carcinoma (OSCC), presenting molecular and/or histological similar alterations. The divergences about the malignization potential of OEDs and the role of inflammation in this process make hard the early diagnosis and evaluation of OSCCs aggressiveness. Thus, it became the goal of this study to evaluate the role of inflammation in oral carcinogenesis and tumoral aggressiveness. For this purpose a morphological study was performed in 20 OED cases and 40 OSCC cases to detect the malignization potential of OEDs and the histologic malignancy grading (HMG) of OSCCs, analyzing superficial masses for dismorphism evaluation and the invasive front for evaluation of tumoral growing; and immunohistochemical, using anti-CD8, anti-FOXP3, anti-TGFβ, anti-TNFα and anti-NF-кB antibodies, comparing their with the types lesion, histological degree and intensity of the inflammatory infiltrate. The results were statistically significant for the parameters: cell maturity (p=0,0001), masses presence (p=0,038) and dismorphism (p=0,037), when associated to HMG. To compare the expression of the markers with the types lesion, a significantly higher expression of CD8 (p=0,001) and NF-кB (p=0,002) in the OED, and also a smaller expression of the epithelial TGFβ in the severe OEDs (p=0,011), without significant expression between OSCC degrees. By relating the expression of the studied markers with the inflammatory infiltrate intensity, a positive relation was observed with: inflammatory TNFα(p=0,003), epithelial TNFα and NF-кB (p=0,051 and p=0,004), in OEDs; and with CD8 (p=0,021) and TNFα (p=0,015) in conjunctive OSCCs; and a negative relation with epithelial TNFα (p=0,034) in OSCCs. No significant relation was found between FOXP3 with any of the studied variables. These findings lead to the conclusion that, the study of the invasive front is as important as the study of superficial masses for the evaluation of tumoral aggressiveness; the intensity of the inflammatory infiltrate has no use as a parameter for prognostic evaluation of OSCC in routine exams, but, the molecular events detected in this study may be necessary to give basis for determining the malignant potential in OEDs and aggressiveness in OSCCs

Avaliacao de polimorfismos nos genes XPD e XRCC3 em carcinomas orais de celulas escamosas

Oral squamous cell carcinoma (OSCC) is an important cause of morbidity and mortality worldwide despite recent advances in treatment. There are several studies aiming to find markers that may improve the assessment of this disease prognosis. Studies about genetic polymorphisms have gained prominence due to their influence on individual susceptibility to cancer development. The aim of this study was to evaluate the association between the frequency of polymorphisms XPD Lys751Gln and XRCC3 Thr241Met and clinicopathological features of OSCC cases, including age, sex, presence or absence of metastases, and histological grading of malignancy according to Bryne (1998). Sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). OSCC cases were classified as low or high grade. DNA samples were previously extracted from paraffin blocks. Genotypes for each case were determined through PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism). Results were analyzed by Fisher s exact test and Chi-square test and the odds ratio was calculated considering p < 0.05 to indicate statistical significance. For XPD, Lys/Gln genotype was more common in IFHs (n=28; 70%) than in OSCCs (n=24; 44.4%) (OR: 0.3; p<0.05). Frequency of Gln allele was higher in high-grade lesions when compared to low grade lesions (0.48 and 0.21, respectively) (OR: 3.4; p<0.05). For XRCC3, Met allele was more common in OSCC than in IFH (0.49 and 0.35, respectively) (OR: 2.6; p<0.05). Met/Met genotype was associated with presence of metastases (OR: 8.1; p<0.05). There was no statistically significant association between the genotypes and the age or sex of patients. In the present sample, the higher frequency of XPD Gln allele in IFH reveals a possible protective role of this variant against the development of OSCC. However, its association with high-grade lesions indicates that this allele could influence the tumor progression after the neoplasia development. The presence of XRCC3 Met allele, in turn, seems to contribute to the development of OSCC and metastases

Correlation of the expression of human kallikrein-related peptidases 4 and 7 with the prognosis in oral squamous cell carcinoma

Background Very few articles have been written about the expression of kallikreins (KLK4 and KLK7) in oral cancers. Therefore, the purpose of this study was to examine and report on their prognostic potential. Methods Eighty archival blocks of primary oral cancers were sectioned and stained for KLK4 and KLK7 by immunohistochemistry. The percentage and the intensity of malignant keratinocyte staining were correlated with patient survival using Cox regression analysis. Results Both kallikreins were expressed strongly in the majority of tumor cells in 68 of 80 cases: these were mostly moderately or poorly differentiated neoplasms. Staining was particularly intense at the infiltrating front. Patients with intense staining had significantly shorter overall survival (p < .05). Conclusion This is the first observation on the patient survival influenced by kallikrein expression in oral carcinoma. The findings are consistent with those for carcinomas at other sites, in particular the prostate and ovary. KLK4 and/or KLK7 immunohistochemistry seems to have diagnostic and prognostic potential in this disease.

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75(NTR) in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75NTR was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75NTR, CD24 antigens and ALDH activity (ALDEFLUOR® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Evaluation of regional and distant metastases in head and neck squamous cell carcinoma patients with special reference to the assessment of regional lymph nodes in oral cancer

For optimal treatment planning, a thorough assessment of the metastatic status of mucosal squamous cell carcinoma of the head and neck (HNSCC) is required. Current imaging methods do not allow the recognition of all patients with metastatic disease. Therefore, elective treatment of the cervical lymph nodes is usually given to patients in whom the risk of subclinical metastasis is estimated to exceed 15-20%. The objective of this study was to improve the pre-treatment evaluation of patients diagnosed with HNSCC. Particularly, we aimed at improving the identification of patients who will benefit from elective neck treatment. Computed tomography (CT) of the chest and abdomen was performed prospectively for 100 patients diagnosed with HNSCC. The findings were analysed to clarify the indications for this examination in this patient group. CT of the chest influenced the treatment approach in 3% of patients, while CT of the abdomen did not reveal any significant findings. Our results suggest that CT of the chest and abdomen is not indicated routinely for patients with newly diagnosed HNSCC but can be considered in selected cases. Retrospective analysis of 80 patients treated for early stage squamous cell carcinoma of the oral tongue was performed to investigate the potential benefits of elective neck treatment and to examine whether histopathological features of the primary tumour could be used in the prediction of occult metastases, local recurrence, or/and poor survival. Patients who had received elective neck treatment had significantly fewer cervical recurrences during the follow-up when compared to those who only had close observation of the cervical lymph nodes. Elective neck treatment did not result in survival benefit, however. Of the histopathological parameters examined, depth of infiltration and pT-category (representing tumour diameter) predicted occult cervical metastasis, but only the pT-category predicted local recurrence. Depth of infiltration can be used in the identification of at risk patients but no clear cut-off value separating high-risk and low-risk patients was found. None of the histopathological parameters examined predicted survival. Sentinel lymph node (SLN) biopsy was studied as a means of diagnosing patients with subclinical cervical metastases. SLN biopsy was applied to 46 patients who underwent elective neck dissection for oral squamous cell carcinoma. In addition, SLN biopsy was applied to 13 patients with small oral cavity tumours who were not intended to undergo elective neck dissection because of low risk of occult metastasis. The sensitivity of SLN biopsy for finding subclinical cervical metastases was found to be 67%, when SLN status was compared to the metastatic status of the rest of the neck dissection specimen. Of the patients not planned to have elective neck dissection, SLN biopsy revealed cervical metastasis in 15% of the patients. Our results suggest that SLN biopsy can not yet entirely replace elective neck dissection in the treatment of oral cancer, but it seems beneficial for patients with low risk of metastasis who are not intended for elective neck treatment according to current treatment protocols.

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.