Yes / Background - Previous studies have demonstrated that a number of HOX genes, a family of transcription factors with key roles in early development, are up-regulated in head and neck squamous cell carcinoma (HNSCC) and other cancers. The loci of several Homeobox (HOX) genes also contain microRNAs (miRs), including miR-196a. Methods - Global miR expression and expression of all 39 HOX genes in normal oral keratinocytes (NOKs), oral pre-malignant (OPM) and HNSCC cells was assessed by expression microarray and qPCR and in tissues by immunohistochemistry (IHC) and qPCR of laser microdissected (LCM) tissues. Expression of miR196a and HOXB9 was reduced using anti-miR-196a and siRNA, respectively. Expression microarray profiles of anti-miR196a and pre-miR196a transfected cells were compared to parental cells in order to identify novel targets of miR- 196a. Putative miR196a targets were validated by qPCR and were confirmed as binding to the 3’UTR of miR196a by a dual luciferase reporter assay combined with mutational analysis of the miR-196a binding site. Results - miR-196a and HOXB9 are highly expressed in HNSCC compared to NOKs, a pattern also seen in HNSCC tissues by HOXB9 IHC and qPCR of miR-196a in LCM tissue. Knock-down of miR-196a expression decreased HNSCC cell migration, invasion and adhesion to fibronectin, but had no effect on proliferation. Furthermore, knock-down of HOXB9 expression decreased migration, invasion and proliferation but did not alter adhesion. We identified a novel primary mRNA transcript containing HOXB9 and miR196a-1 as predicted from in-silico analysis. Expression array analysis identified a number of miR196a targets, including MAMDC2 and HOXC8. We confirmed that MAMDC2 is a novel miR-196a target using a dual luciferase reporter assay with the effect abolished on mutation of the binding site. Conclusions - These results show that miR-196a and HOXB9 are overexpressed, perhaps co-ordinately, as HNSCC develops and exert a pro-tumourigenic phenotype in HNSCC and OPM cells.

Cancer is a major cause of morbidity and mortality with increasing incidence worldwide. Early detection of cancers and better treatments would improve the outcome for patients. The overall 5-year survival rates of head and neck squamous cell carcinoma have not improved in the past several decades due to diagnosis at advanced stages and recurrent disease. Early detection and improved chemotherapy drugs are two key areas that are required to help to improve the prognosis for this disease. This thesis focuses on the enzyme telomerase which is known to contribute to one of the hallmarks of cancer (immortality). Elevated telomerase activity has been observed in the majority of cancer cells but not in most normal human cells so there is an opportunity to use telomerase as a biomarker for disease. This first part of this study assessed telomerase activity in saliva and tissues of head and neck squamous cell cancer patients. The Telomerase PCR-ELISA kit was used to assess telomerase activity in the saliva of patients with confirmed oral carcinomas and its expression was analysed in paraffin embedded tissue using immunohistochemistry (IHC). Whilst telomerase was detected in cell lines, no telomerase activity was detected in saliva samples from patients but was detectable in IHC specimens. The second part of the study focused on the pharmacological evaluation of a series of small molecule G–quadruplex DNA binding agents as potential telomerase inhibitors. A total of 19 telomerase inhibitors were identified but of these, only 4 were specific inhibitors of telomerase. These compounds also caused toxicity to cell lines following a 2 hour drug exposure at doses that also inhibit telomerase activity. Further studies are required to explore these compounds further. In conclusion, the results of this study have demonstrated that detection of telomerase activity I the saliva of patients with oral cancers is unlikely to be useful in terms of detecting oral cancers before symptoms of the disease are clinically manifest. A series of novel and specific inhibitors of telomerase have been identified and further studies are required to develop these compounds further.

Squamous cell carcinoma (SCC), the most common oral neoplasm of cats, demonstrates aggressive local invasion and has a poor prognosis. In humans, mutation of the p53 gene, crucial in cell cycle arrest and induction of apoptosis in damaged cells, is common in neoplasms. Survivin, an inhibitor of apoptosis, is frequently overexpressed in many types of human cancer. Studies suggest that wild-type p53 inhibits survivin expression, while mutated p53 does not. The purposes of this study included immunohistochemical examination of survivin and p53 expression in feline oral SCC and determination of a correlation between p53 mutation and survivin overexpression, as well as comparison with survival time. Survivin expression was noted in 80% (24/30) of cases, while 43.3% (13/30) of cases were positive for p53. No statistically significant correlation was noted between p53 and survivin expression, even when corrected for age, breed, and sex; and survival time was not affected.

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Yes / Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies. / Malaysia Ministry of Education (Fundamental Research Grant Scheme, FRGS Vot. 1482 and IGSP Vot. 679).