Development of 3D tissue-engineered larynx using nanocomposite POSS-PCU material and stem cells

Background: Loss of laryngeal function severely affects the quality of life. To date, there are no optimised implants for laryngeal replacement or reconstruction; hence, it is still considered an unmet clinical need. The aim of this research is to develop a 3D scaffold made from POSS-PCU materials. This scaffold could then be assessed the biocompatibility with human primary bronchial epithelial cells (HBEC) and bone-marrow mesenchymal stem cells (BM-MSC) to produce tissue-engineered artificial larynx. Methods: 3D laryngeal scaffolds were fabricated from polyhedral oligomeric silsesquioxane – poly(carbonate-urea)urethane (POSS-PCU). The scaffolds were designed to be microporous with sodium bicarbonate (NaHCO3) particle size of 25 - 40 µm at different concentrations. This procedure created an interconnected microporous network and can be coated on the outer surface of casted POSS-PCU to create the 3D laryngeal frameworks. The materials were fully characterised and optimised for biocompatibility and voice function. HBEC and BM-MSC were seeded onto scaffolds and observed the viability as well as the characteristics after differentiation on the material such as morphology, and specific protein expressions via staining. Results: Comparison studies of mechanical strength showed no significant difference between porcine epithelial tissue and microporous POSS-PCU (P > 0.05), while the mechanical strength of the non-porous POSS-PCU was higher than the microporous samples and native epithelial tissue and cartilage (P 0.05). The material also showed the significant protein adsorption and enhanced cell attachment and growth in all samples with ethanol rinse. Primary HBEC and BM-MSC had significant growth on 3D porous scaffolds over 14 days (P < 0.05). Furthermore, the scaffold supported the differentiation of primary HBECs using air-liquid interface culture to develop mature epithelial cells with pseudostratified layer and migration into the POSS-PCU materials. SEM and immunofluorescence staining revealed the specific epithelial characteristics which were keratin 5 in the basal layer, keratin 18 on the top layer, epithelial tight junction, ciliated cells and mucin5AC production. BM-MSC also presented the growth and the differentiation on the material. Chondrogenic lineage properties had been observed through the change in cell morphology and the increased production of cartilaginous-like ECM such as sulphatedglycosaminoglycan (sGAG) and collagen. Conclusion: It had been demonstrated that the 3D scaffold made from POSS-PCU promotes cell adhesion, proliferation and differentiation with mature epithelial features as well as chondrogenic properties. This scaffold holds great promise to develop a tissueengineer-based larynx for laryngotracheal replacements

Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin

Increased immunoexpression of trefoil factors in salivary gland tumors

OBJECTIVE: Very little is known about the role of trefoil factors (TFFs) in salivary gland tumors, and TFF immunoexpression has never been investigated in such tumors. The aim of this study was to evaluate TFF immunoexpression in benign and malignant salivary gland tumors. MATERIALS AND METHODS: Benign (n = 25) and malignant (n = 25) salivary gland tumor specimens were included in this study, using mucocele (n = 25) specimens as a control group. Immunohistochemical staining was performed to evaluate the expression of TFFs (TFF1, TFF2, and TFF3) by semiquantitative means. RESULTS: Expression of TFF1, TFF2, and TFF3 was significantly increased in benign (p = 0.001, p = 0.005, p < 0.001, respectively) and malignant (p < 0.001, p < 0.001, p < 0.001, respectively) groups as compared with the control group. Patterns of co-expression between TFF1/TFF2, TFF2/TFF3, and TFF1/TFF3 were different among the three groups. CONCLUSIONS: The present study provided new information showing that all TFFs were significantly increased in benign and malignant salivary gland tumors, and overexpression of TFFs could be associated with neoplastic transformation in salivary gland tissues. CLINICAL RELEVANCE: Overexpression of TFFs may be useful as biomarkers in terms of differential diagnosis between salivary gland tumors and other oral neoplasms for which clinical manifestations are indistinguishable

Increased immunoexpression of trefoil factors in salivary gland tumors

OBJECTIVE: Very little is known about the role of trefoil factors (TFFs) in salivary gland tumors, and TFF immunoexpression has never been investigated in such tumors. The aim of this study was to evaluate TFF immunoexpression in benign and malignant salivary gland tumors. MATERIALS AND METHODS: Benign (n = 25) and malignant (n = 25) salivary gland tumor specimens were included in this study, using mucocele (n = 25) specimens as a control group. Immunohistochemical staining was performed to evaluate the expression of TFFs (TFF1, TFF2, and TFF3) by semiquantitative means. RESULTS: Expression of TFF1, TFF2, and TFF3 was significantly increased in benign (p = 0.001, p = 0.005, p < 0.001, respectively) and malignant (p < 0.001, p < 0.001, p < 0.001, respectively) groups as compared with the control group. Patterns of co-expression between TFF1/TFF2, TFF2/TFF3, and TFF1/TFF3 were different among the three groups. CONCLUSIONS: The present study provided new information showing that all TFFs were significantly increased in benign and malignant salivary gland tumors, and overexpression of TFFs could be associated with neoplastic transformation in salivary gland tissues. CLINICAL RELEVANCE: Overexpression of TFFs may be useful as biomarkers in terms of differential diagnosis between salivary gland tumors and other oral neoplasms for which clinical manifestations are indistinguishable

Specific isoforms of p73 control the induction of cell death induced by the viral proteins, E1A or apoptin

A member of the p53 family, p73, has several isoforms and differentially regulates transcription of genes involved in the control of the cell cycle and apoptosis. We have previously shown efficient and p53-independent, tumor-specific cell death induced by the viral proteins E1A and Apoptin. Here, we demonstrate that the induction of apoptosis by these viral proteins involves activation of TAp73. Both E1A and Apoptin induced expression of endogenous TAp73 and the p53/p73 BH3-only pro-apoptotic target, PUMA, independently of the p53 function. Furthermore, exogenous expression of TAp73 isoforms, particularly TAp73β, sensitized cells to killing by both E1A and Apoptin, while expression of ΔNp73α blocked this activity. Besides, knockout of the p73 regulator, c-Abl, attenuated E1A-induced apoptosis. In accordance with the role of p73 in apoptosis induced by these viral proteins, overexpression of TAp73β strongly induced apoptosis in p53-deficient cancer cells in vitro and in HNSCC xenografts. Using a doxycycline-inducible system, we provide evidence for target selectivity and significant differences in protein stability for specific p73 isoforms, suggesting a diverse and pivotal role for p73 in response to various genotoxic agents. Collectively, our data show that in the absence of the p53 function, viral proteins E1A and Apoptin utilize the p73 pathway to induce efficient tumor cell death

PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers

Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic ΔNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73β, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73β-induced EGFR suppression and apoptosis. Expression of TAp73β efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to) have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells., remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis