1,721,124 research outputs found
Urinary biomarkers for the prediction of acute kidney injury in major abdominal surgery
Full text linkAcute Kidney Injury (AKI) can affect one in five major abdominal surgery (MAS) patients. AKI is defined by the ‘Kidney Disease: Improving Global Outcomes’ criteria, based on rises in serum creatinine and low urine output. This approach has limitations that significantly delay identification.Novel biomarker tests have been investigated that aim to identify AKI rapidly, performing strongly in cardiac, transplant and vascular surgery where AKI rates approach 50%. A systematic review conducted to inform this thesis identified six papers examining biomarkers in MAS: methodological concerns were common throughout. Biomarker utility in MAS is thus unknown.This observational study investigated the diagnostic performance of urinary biomarkers in 488 MAS patients. Biomarkers scrutinised were Neutrophil Gelatinase associated Lipocalin (NGAL), Insulin like Growth Factor Binding Protein-7 combined with Tissue Inhibitor of Metalloproteinases-2 (NC), Kidney Injury Molecule-1(KIM-1) and Dickkopf-3 (DKK-3). Concentrations were measured four hours post-operatively and assessed using area under the receiver operator curve analysis (AUROC).AKI by serum creatinine (AKISCr) occurred in 8.8% of patients, and by urine output (AKIUO) in 49.6%. NGAL had the highest AUROC for AKISCr (0.74), followed by KIM-1(0.68) and NC (0.66). Performance improved with worsening AKI. AUROC values were below 0.65 for nearly all stages of AKIUO.Logistic regression analysis revealed increasing age, body mass Index, operation duration, American Association of Anaesthesiologists grade, and male sex to be strongly associated with the development of AKISCr. A risk prediction model incorporating these factors was created and returned an AUROC of 0.8. With the best performing biomarker added (NGAL), the AUROC was 0.82.This is the largest study interrogating AKI biomarkers in MAS, identifying a biomarker test and risk prediction model that perform well for AKISCr. An interventional study has been proposed that randomises biomarker positive MAS patients to an AKI ‘bundle’ or standard care. This may demonstrate whether early AKI identification with biomarkers translates into improved outcomes
Using HOX Transcription Factors as Novel Diagnostic Biomarkers and HOX-PBX Interactions as Therapeutic Targets in Breast Cancer
Full text linkTriple Negative Breast Cancer (TNBC) represents 10-15% of all breast cancers, is characterised by its lack of hormone and HER2 receptor expression and exist as clinically high grade and aggressive tumours with poor prognosis. A major challenge of these tumours is the lack of effective targeted therapies and the resistance to conventional therapies. Homeobox (HOX) genes are a family of homeodomain-containing transcription factors, which are primarily known for establishing and maintaining the identity and fate of cells and tissues during normal embryogenesis and organ development. Recent studies have highlighted HOX genes to be dysregulated in most cancers including breast, ovarian, prostate, colorectal and melanoma, and possess an established role in driving key hallmarks in carcinogenesis, including the regulation of the cell cycle, apoptosis, angiogenesis, and metastasis. In this study, the HOX expression profiles between TNBC and all other breast cancer subtypes was evaluated relative to normal HOX expression and showed TNBC tumours possess unique and specific HOX expression profiles and were indicative of prognosis. Given their cancer-specific expression, targeting HOX proteins for cancer therapy would potentially provide a novel therapeutic option, as yet unexploited. Rather than targeting individual HOX genes, which is ineffective due to the functional redundancy of HOX genes, targeting multiple HOX proteins post-translationally becomes feasible by inhibiting the interaction between HOX and their PBX co-factors by disrupting protein-protein interactions.In this study, we evaluated the anti-tumour efficacy of a novel inhibitor of HOX protein function, HTL-001, which antagonizes the interactions between HOX and PBX proteins, thus preventing the binding of this complex to DNA to promote tumour growth. HTL-001 is a synthetic peptide comprised of a hexapeptide sequence, which resembles the interaction site between HOX proteins of paralogs 1-9 and PBX proteins. Targeting HOX/PBX dimers with HTL-001, induced apoptotic cell death in all breast cancer cell lines including MCF-7, ZR-75-1, MDA-MB-231, BT-20 and SK-BR-3, with the highest sensitivity seen in brain seeking MDA-MB-231-BR cells. HTL-001 also showed high synergistic anti-cancer effects in TNBC cell lines when combined with conventional chemotherapies and significantly translated these effects in vivo with significant inhibition of tumour growth by HTL-001 alone and in combination with Paclitaxel. The mechanism of action of HTL-001 was explored and the downstream effects on apoptosis was examined in detail. Disruption of HOX-PBX dimers by HTL-001 resulted in the upregulated expression of key apoptotic and anti-survival proteins that form an interconnecting network of signalling pathways initiated by ROS mediated ER stress which causes calcium influx and calpain activation to regulate caspase-independent activation of AIF, and release of immunogenic DAMPs. Mechanisms of synergy were evaluated to additionally show the main mechanism of synergistic apoptosis results from activating HTL-001 mediated signalling pathways and enhancing these by a chemotherapeutic agent. Resistance to HTL-001 was found to be mediated by autophagy and can be overcome by autophagy inhibitors. HTL-001 is currently the only therapy which targets HOX gene dysregulation and in doing so, may help address the current urgent unmet therapeutic need in TNBC through its unique mechanism of action and high levels of synergy with conventional anti-cancer agents
Investigating engrailed-2 (EN2) as a therapeutic target in prostate cancer.
No full textEngrailed-2 (EN2) is a transcription factor involved in development, where it has multiple functions and is expressed in a caudal-to-rostral gradient in the midbrain. Its mRNA and protein expression are absent from most adult cells, but are switched back on in cancer cells. Although transcription factors are usually located in the nucleus, a number of previous reports have indicated that EN2 protein could be in the cell membrane and on the cell surface on tumour samples, from where some at least might be released, as EN2 has been found in the urine of prostate cancer patients - a more readily obtainable clinical sample than tumour biopsies. In this study EN2 protein is definitively shown to be on the membrane of prostate cancer cells and in the tumour microenvironment. The commercial anti-EN2 antibody was found to be non-specific and we therefore used a tagged version of EN2 to study its cellular distribution and behaviour. This revealed different modes of EN2 protein transport and secretory mechanisms in different cancer cell lines. Live cell imaging further revealed the generation of secretory vesicles from PC3 cells, which are derived from metastatic prostate cancer, but not WPMY-1 cells that are derived from normal prostate fibroblasts. The findings further suggest that EN2 protein switches roles during tumour progression, from a transcriptional regulator to a regulator of protein translation in localised regions of the cytoplasm. The latter mechanism is especially significant as EN2 cellular localisation becomes dysregulated in cancer, becoming widely cytoplasmic and available for packaging into luminal vesicles. The findings also indicate that the translation factor EIF4E is a potential binding partner of EN2, in prostate cancer. The study findings also indicate that a monoclonal antibody-drug conjugate targeting EN2 may not be the most effective method of targeting EN2-expressing cells. A blocking peptide or antibody would be more appropriate to prevent its secretion and transfer, both of which have been shown to be possible mechanisms of tumour progression. Alternatively, because less EN2 is secreted (and hence more is retained) in prostate cancer cell lines with low metastatic potential, such as LnCaP, T cells could be employed to target early-stage prostate cancer. To conclude, cancer cells have seemingly retained the ability to tightly regulate the expression of EN2 protein in a spatial and temporal manner, unlike normal adult cells. EN2 is secreted in large vesicles by cells from more advanced prostate tumours and thus monoclonal antibodies may not be the most effective approach to therapy
Investigating engrailed-2 (EN2) as a therapeutic target in prostate cancer.
Full text linkEngrailed-2 (EN2) is a transcription factor involved in development, where it has multiple functions and is expressed in a caudal-to-rostral gradient in the midbrain. Its mRNA and protein expression are absent from most adult cells, but are switched back on in cancer cells. Although transcription factors are usually located in the nucleus, a number of previous reports have indicated that EN2 protein could be in the cell membrane and on the cell surface on tumour samples, from where some at least might be released, as EN2 has been found in the urine of prostate cancer patients - a more readily obtainable clinical sample than tumour biopsies.
In this study EN2 protein is definitively shown to be on the membrane of prostate cancer cells and in the tumour microenvironment. The commercial anti-EN2 antibody was found to be non-specific and we therefore used a tagged version of EN2 to study its cellular distribution and behaviour. This revealed different modes of EN2 protein transport and secretory mechanisms in different cancer cell lines. Live cell imaging further revealed the generation of secretory vesicles from PC3 cells, which are derived from metastatic prostate cancer, but not WPMY-1 cells that are derived from normal prostate fibroblasts. The findings further suggest that EN2 protein switches roles during tumour progression, from a transcriptional regulator to a regulator of protein translation in localised regions of the cytoplasm. The latter mechanism is especially significant as EN2 cellular localisation becomes dysregulated in cancer, becoming widely cytoplasmic and available for packaging into luminal vesicles. The findings also indicate that the translation factor EIF4E is a potential binding partner of EN2, in prostate cancer.
The study findings also indicate that a monoclonal antibody-drug conjugate targeting EN2 may not be the most effective method of targeting EN2-expressing cells. A blocking peptide or antibody would be more appropriate to prevent its secretion and transfer, both of which have been shown to be possible mechanisms of tumour progression. Alternatively, because less EN2 is secreted (and hence more is retained) in prostate cancer cell lines with low metastatic potential, such as LnCaP, T cells could be employed to target early-stage prostate cancer.
To conclude, cancer cells have seemingly retained the ability to tightly regulate the expression of EN2 protein in a spatial and temporal manner, unlike normal adult cells. EN2 is secreted in large vesicles by cells from more advanced prostate tumours and thus monoclonal antibodies may not be the most effective approach to therapy
Measles virus causes immunogenic cell death in human melanoma
No full textNoOncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response
Profiling and targeting HOX-PBX dimers in adult and paediatric glioblastoma as a novel therapy.
Full text linkHOX genes encode a family of transcription factors that play an essential role in embryonic patterning during foetal development. These genes are reported to be aberrantly expressed in numerous cancers, including glioblastoma (GBM). Previous research indicates that HOX genes are overexpressed in GBM tumours compared to normal human astrocytes (NHA). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE members Pre-B-cell leukaemia homeobox (PBX) 1-4, bind anterior HOX proteins, facilitating cellular entry, while also limiting their degradation. HXR9, a small hexapeptide drug, has previously been shown to inhibit HOX-PBX dimers, causing rapid cell death in numerous solid and haemopoietic malignancies both in vitro and in vivo. HTL00-1 is a 2nd generation peptide based on HXR9, while ICT9119 is a small molecular inhibitor with the same binding site as the peptide inhibitors.
We found clear evidence of overexpression of both HOX and TALE genes, in both adult and paediatric GBM cell lines. A further elevation in expression of these genes was visible in GBM cancer stem cells (CSCs). Normal adult brain tissue expressed low to undetectable levels of HOX genes, while paediatric brains expressed high levels between 0-4 years of age, before dramatically reducing from ages 5 onwards. HOX-PBX inhibitors are shown to be both cytostatic and cytotoxic when used to treat adult and paediatric GBM cells both in vitro and in vivo. GBM CSCs were more sensitive to HOX-PBX inhibition, alluding to their increased reliance on these genes. HTL00-1 and ICT9119 were shown to provide improved pharmacological effects compared to their predecessor HXR9. All HOX-PBX inhibitors cause apoptosis through induction of Fos Proto-Oncogene AP-1 Transcription Factor Subunit (C-Fos) expression, which results in the dimerization of C-Fos and Jun Proto-Oncogene AP-1 Transcription Factor Subunit (C-Jun) to form the Activator protein 1 (AP-1) complex. This directly and indirectly represses B-cell lymphoma 2 (Bcl-2) expression leading to the release, and subsequent cleavage of caspase 3 and 7. This triggers the caspase cascade that concludes with cellular apoptosis.
Radiotherapy (RT), combined with ICT9119 treatment proved highly synergistic in terms of cell kill when applied in combination when cancer cells were exposed to RT 24 hours prior to ICT9119. When ICT9119 was delivered 24 hours prior to RT antagonistic effects were observed. Attempts to identify the molecular mechanisms behind differing sequential treatment outcomes proved inconclusive. However, our work did suggest a mixture of cell cycle sequestration, C-Jun induction and the opposing effects of dual specific phosphatase 1 (DUSP1) and Mitogen-activated protein kinases (MAPK) were at least partly involved.
This study highlights the important role of HOX genes in paediatric and adult gliomas, the potential utility of HOX/TALE gene expression in the identification of CSC population in patients and potential for HOX-PBX inhibition as a highly targeted effective treatment for adult and paediatric GBM patients. This treatment can be administered in the form of a peptide, as in HXR9 or HTL00-1, or as a small molecular agent in the form of ICT9119, either as single agents, or in combination with RT. HOX-PBX disruption as described in this thesis could potentially be moved rapidly into the clinic, in an area of very high unmet need
Evaluation of the effects of oncolytic vaccinia virus on colorectal liver metastases in cell lines and in organotypic cultures.
Full text linkVaccinia virus is one of several live viruses currently being evaluated in clinical trials as cancer therapies. One oncolytic virus, T-VEC, is already in clinical use and is NICE approved for the treatment of melanoma. We have investigated a genetically modified, Copenhagen strain vaccinia virus encoding the fusion suicide gene (FCU1) that is able to convert non-toxic fluorocytosine into the active compound fluorouracil. The mode of cell death attributed to vaccinia virus is variable and multiple modes have been implicated based on tumour type. Through our work with colorectal cancer cell lines we have established that this form of oncolytic vaccinia virus induces cell death through a predominant picture of apoptosis. Furthermore, assessment of its immunogenicity has found there to be an increase in markers of immunogenic cell death following infection with the virus. We have developed an organotypic culture system in our laboratory which enables us to assess oncolytic viruses on ex vivo tissue. Treatment of organotypic cultures from colorectal liver metastases have shown that vaccinia virus successfully infects and replicates in the tumour tissue. A dose-response relationship was observed with the highest doses of virus exerting the most effect on tumour tissue. Assessment of the pro-drug activation system through the conversion of 5-fluorocytosine to 5-fluorouracil in supernatant showed a high percentage conversion, whilst immunohistochemical staining did show evidence of an improved effect with the pro-drug system compared with virus alone. We have evaluated the combination of cavitational ultrasound and sulphur hexafluoride microbubbles to enhance infection with oncolytic virus. Our results have shown that this method enhances infection of organotypic cultures treated with oncolytic vaccinia virus. This has enabled the use of a lower dose of virus whilst maintaining the same effects in tissue as that of virus at higher doses. This finding has translational implications that may enhance the efficacy of systemically administered oncolytic vaccinia virus
Coxsackievirus A21 used as an oncolytic immunotherapy for cancer.
Full text linkOncolytic viruses selectively target and kill cancer cells by direct lysis and by stimulating a tumour antigen specific immune response. These promising therapeutic agents target multiple cancers and one such agent, T-Vec (Talimogene laherparepvec) has been licensed for treatment of malignant melanoma. We investigated the potential of Coxsackievirus A21 (CVA21) as a treatment for bladder and pancreatic cancers. The human bladder cancer cell-lines were tested and cytolytic ability of CVA21 depended on the expression of the intercellular adhesion molecule -1 (ICAM-1) viral entry receptor which was increased by Mitomycin C, this led to enhanced viral replication and cell death. CVA21 oncolysis induced immunogenic apoptosis as characterised by an increase in expression of ICD markers and apoptosis markers. In addition, MB49/ICAM-1 bladder cancer cells undergoing CVA21-induced ICD led to MB49 tumour rejection in a syngeneic murine bladder cancer model, protection was provided by CD4+ T cells. We evaluated the mechanism of resistance to CVA21 infection in human pancreatic cancer cell-lines. Despite the presence of high ICAM-1, BxPC-3 cell-line was resistant to CVA21 oncolysis and no viral protein was detected. As no genetic mutation was detected in the ICAM-1, an investigation of the mutational background revealed a lack of KRAS mutations in the BxPC-3 cell-line. This lack of KRAS mutation has previously shown to be involved in resistance to virus susceptibility. The therapeutic potential of CVA21 was evaluated by a murine bladder cancer model. Although, CVA21 intratumoural administration delayed tumour growth for a short time. There were limitations such as unrestricted tumour growth and short life-span of the model due to which the evaluation of virus induced immune targets for potential combinational approaches was not possible. Although, CVA21 has shown its benefit as monotherapy both in cell-lines and in in vivo model systems emerging data shows enhanced treatment efficacy using oncolytic viruses in combination with other immune modulatory agents
Dynamic changes in cervical cancer.
Full text linkLocally advanced cervical cancer is treated with chemoradiotherapy. The 5 year survival for stage II and III disease ranges from 40-60% and therefore there is scope for improvement of treatment either using dose escalation or adding in additional therapeutic agents. However, the burden of toxicity following chemoradiotherapy is not insignificant and therefore whilst trying to improve the cure rate, it is also important to reduce the dose to the organs at risk.
This thesis looks at methods of increasing the dose delivered to the tumour whilst reducing the dose to the organs at risk. The methods used to achieve this are the use of a bladder scanner to reduce internal organ movement, the use of a plan of the day strategy, the use of interstitial needles for brachytherapy and the use of texture analysis to predict those patients who are not going to achieve a complete response to chemoradiotherapy.
The final chapter starts to explore the question of whether immunotherapy in the form of immune checkpoint blockade drugs could potentially be of benefit in cervical cancer. The tumour microenvironment was assessed before and during chemoradiotherapy by looking at tumour infiltrating lymphocyte populations and the expression of PD-1 and PD-L1. These were also studied in the peripheral blood
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