Browsing by Author "Neves, Ana Raquel Bastos"
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- Cancer gene therapyPublication . Neves, Ana Raquel Bastos; Costa, Diana Rita Barata; Sousa, Ângela Maria Almeida deCancer is one of the major causes of morbidity and mortality worldwide. It involves genetic changes that affect a variety of genes, namely tumour suppressor genes. The traditional approaches for cancer treatment include chemotherapy, surgery and radiation. Chemotherapy represents the main choice of treatment in most cases, however, traditional therapies seemed unvalued to fight against metastasis, recurrence of tumour, and the treatment of advanced cancer. Therefore, new strategies need to be developed to increase therapy efficacy. Gene therapy has brought a promising and unique approach to medicine because of its wide application in the treatment of various diseases, including hereditary diseases to acquired (infection or cancer) pathologies. p53 suppressor gene mutation or degradation is found in more than 50% of human cancers, therefore, gene therapy protocols focussed on the restauration of p53 protein are a priority in this field. For gene therapy viability in a clinical setting, the development of an efficient gene delivery system is imperative. The conception of delivery systems based on cell penetrating peptides represents an incredible asset and may deeply contribute for the evolution of cancer therapy. In this context, a new system for p53 encoding plasmid DNA (pDNA) delivery based on RALA peptide was designed and developed in order to produce a suitable intracellular delivery platform capable of gene delivery and restoration of p53 levels within the cancer cells. These carriers were characterized in terms of morphology, size, surface charge, loading and encapsulation efficiency and the fine structure was analyzed by Fourier-transformed infrared (FTIR) spectroscopy. The results showed that formed nanoparticles are suitable for cell uptake, internalization and gene release. Furthermore, stability studies demonstrated that RALA is capable of protect encapsulated DNA from serum nucleases and MTT assay showed that these systems are biocompatible. Confocal microscopy and live cell imaging experiments confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allowed the detection of mRNA transcripts by RT-PCR and p53 protein expression by Western Blot. An ELISA kit assay quantified the produced protein levels. From these progresses, apoptosis in cancer cells was investigated. Caspase 3 activation was monitored by means of colorimetric assay and TUNEL assay enabled to confirm nuclear DNA fragmentation post transfection with the carriers. Lastly, a western blot assay with BAX antibody permitted to figure out which apoptosis pathway is responsible for cancer cells death. Taken together, the presented results revealed that the RALA/pDNA vector seems to be suitable as an innovative platform for p53 mediated cancer gene therapy.