http://hdl.handle.net/10468/937 Thu, 19 Oct 2017 03:37:26 GMT 2017-10-19T03:37:26Z http://hdl.handle.net/10468/4835 Designer bacteria as intratumoural enzyme biofactories Lehouritis, Panos; Hogan, Glenn; Tangney, Mark Bacterial-directed enzyme prodrug therapy (BDEPT) is an emerging form of treatment for cancer. It is a biphasic variant of gene therapy in which a bacterium, armed with an enzyme that can convert an inert prodrug into a cytotoxic compound, induces tumour cell death following tumour-specific prodrug activation. BDEPT combines the innate ability of bacteria to selectively proliferate in tumours, with the capacity of prodrugs to undergo contained, compartmentalised conversion into active metabolites in vivo. Although BDEPT has undergone clinical testing, it has received limited clinical exposure, and has yet to achieve regulatory approval. In this article, we review BDEPT from the system designer's perspective, and provide detailed commentary on how the designer should strategize its development de novo. We report on contemporary advancements in this field which aim to enhance BDEPT in terms of safety and efficacy. Finally, we discuss clinical and regulatory barriers facing BDEPT, and propose promising approaches through which these hurdles may best be tackled. Tue, 12 Sep 2017 00:00:00 GMT http://hdl.handle.net/10468/4835 2017-09-12T00:00:00Z http://hdl.handle.net/10468/3763 UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells Falvey, Chloe M.; O'Donovan, Tracey R.; El-Mashad, Shereen M.; Nyhan, Michelle J.; O'Reilly, Seamus; McKenna, Sharon L. Esophageal cancer remains a poor prognosis cancer due to advanced stage of presentation and drug resistant disease. To understand the molecular mechanisms influencing response to chemotherapy, we examined genes that are differentially expressed between drug sensitive, apoptosis competent esophageal cancer cells (OE21, OE33, FLO-1) and those which are more resistant and do not exhibit apoptosis (KYSE450 and OE19). Members of the ISG15 (ubiquitin-like) protein modification pathway, including UBE2L6 and ISG15, were found to be more highly expressed in the drug sensitive cell lines. In this study, we evaluated the contribution of these proteins to the response of drug sensitive cells. Depletion of UBE2L6 or ISG15 with siRNA did not influence caspase-3 activation or nuclear fragmentation following treatment with 5-fluorouracil (5-FU). We assessed autophagy by analysis of LC3II expression and Cyto-ID staining. Depletion of either ISG15 or UBE2L6 resulted in enhanced endogenous autophagic flux. An increase in autophagic flux was also observed following treatment with cytotoxic drugs (5-FU, rapamycin). In ISG15 depleted cells, this increase in autophagy was associated with improved recovery of drug treated cells. In contrast, UBE2L6 depleted cells, did not show enhanced recovery. UBE2L6 may therefore influence additional targets that limit the pro-survival effect of ISG15 depletion. These data identify UBE2L6 and ISG15 as novel inhibitors of autophagy, with the potential to influence chemosensitivity in esophageal cancer cells. Wed, 08 Feb 2017 00:00:00 GMT http://hdl.handle.net/10468/3763 2017-02-08T00:00:00Z http://hdl.handle.net/10468/4791 Intratumoural production of TNF alpha by bacteria mediates cancer therapy Murphy, Carola; Rettedal, Elizabeth; Lehouritis, Panos; Devoy, Ciaran; Tangney, Mark Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNF alpha) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic E. co/istrain MG1655 was investigated as a tumour targeting system in order to produce TNF alpha specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNF alpha producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNF alpha production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models (CT26 colon, RENCA renal, and TRAMP prostate) as evidenced by tumour volume and survival analyses. A pattern of pro inflammatory cytokine induction was observed in tumours of treated mice vs. controls. Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNF alpha expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours. Sun, 01 Jan 2017 00:00:00 GMT http://hdl.handle.net/10468/4791 2017-01-01T00:00:00Z http://hdl.handle.net/10468/4124 Antitumour responses induced by a cell-based Reovirus vaccine in murine lung and melanoma models Campion, Ciorsdan A.; Soden, Declan; Forde, Patrick F. Background: The ever increasing knowledge in the areas of cell biology, the immune system and the mechanisms of cancer are allowing a new phase of immunotherapy to develop. The aim of cancer vaccination is to activate the host immune system and some success has been observed particularly in the use of the BCG vaccine for bladder cancer as an immunostimulant. Reovirus, an orphan virus, has proven itself as an oncolytic virus in vitro and in vivo. Over 80 % of tumour cell lines have been found to be susceptible to Reovirus infection and it is currently in phase III clinical trials. It has been shown to induce immune responses to tumours with very low toxicities. Methods: In this study, Reovirus was examined in two main approaches in vivo, in mice, using the melanoma B16F10 and Lewis Lung Carcinoma (LLC) models. Initially, mice were treated intratumourally (IT) with Reovirus and the immune responses determined by cytokine analysis. Mice were also vaccinated using a cell-based Reovirus vaccine and subsequently exposed to a tumourigenic dose of cells (B16F10 or LLC). Using the same cell-based Reovirus vaccine, established tumours were treated and subsequent immune responses and virus retrieval investigated. Results: Upregulation of several cytokines was observed following treatment and replication-competent virus was also retrieved from treated tumours. Varying levels of cytokine upregulation were observed and no replication-competent virus was retrieved in vaccine-treated mice. Prolongation of survival and delayed tumour growth were observed in all models and an immune response to Reovirus, either using Reovirus alone or a cell-based vaccine was also observed in all mice. Conclusion: This study provides evidence of immune response to tumours using a cell-based Reovirus vaccine in both tumour models investigated, B16F10 and LLC, cytokine induction was observed with prolongation of survival in almost all cases which may suggest a new method for using Reovirus in the clinic. Wed, 13 Jul 2016 00:00:00 GMT http://hdl.handle.net/10468/4124 2016-07-13T00:00:00Z