Browsing by Author "Pinto, Adriana Resende"
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- Proteomic study of the therapeutic effect of a DNA vector on cervical cancer cellsPublication . Pinto, Adriana Resende; Sousa, Ângela Maria Almeida de; Passarinha, Luís António PaulinoCancer is a major global health problem, accounting for more than 8 million deaths/year worldwide. In particular, cervical cancer is the 3rd most common malignancy and 4th cause of death among women globally, being a persistent High Risk-HPV infection the main factor for cervical cancer development. E6 and E7 HPV oncoproteins are responsible for cancer development by degrading and inhibiting p53 and pRb tumour suppressor proteins, respectively. Moreover, microRNAs (miRs) have been found to regulate tumorigenesis. In fact, miR-375 has the ability to silence HPV E6 and E7 oncoproteins. Gene therapy is a promising strategy to treat acquired diseases and/or genetic disorders, aiming to deliver genetic material into target cells or tissue, expressing it to induce a therapeutic effect. Minicircle DNA (mcDNA) is a new biopharmaceutical product only composed by the eukaryotic transcription unit, improving its safety and therapeutic effect, obtained through intramolecular recombination of the parental plasmid. Thus, this work aims to produce and purify a mcDNA vector encoding primiR-375 and p53 genes to silence E6 and E7 oncoproteins and re-establish p53 and pRb tumour suppressor levels on cervical cancer cells. The purification of mcDNA vectors was performed using size exclusion chromatography sepharose columns (Sephacryl S-1000 SF), showing that for the smallest vector, mcDNAprimiR-375, a 106 mL column allowed the efficient recovery of chromatographic fractions composed only with mcDNA. On the other hand, for the biggest mcDNA vectors, it was necessary to exploit the parameters affecting the sample molecules separation, choosing to use a column with 180 mL of volume, allowing, similarly to the mcDNA-primiR-375 purification, recover fraction only composed with mcDNA. Simultaneously, cytotoxicity assays on human fibroblasts and CaSki cells were performed, confirming that none of the vectors were toxic to the fibroblasts and the mcDNA primiR-375+p53 presented the lower cell viability for CaSki cells. In addition, the proliferation assay results performed on CaSki cells show that number of viable cells decreases for the mcDNA vectors transfected cells, corroborating the cytotoxicity assays results for this cell line. Western-Blot confirmed the re-establishment of tumour suppressor proteins levels after 48h of transfection using mcDNA-p53 and mcDNAprimiR-375+p53. Overall, these results suggest that the use of DNA vectors encoding for more than one therapeutic gene, for example, the mcDNA-primiR-375+p53, allowed to achieve, on in vitro assays, results suggesting a possible faster therapeutic action, thus demonstrating that they may revolutionize their application in targeted therapies.