Cellular and in-vitro models to assess antioxidant activities of seaweed extracts and the potential use of the extracts as ingredients

Seaweeds contain a range of antioxidant compounds such as polyphenols, carotenoids, sulphated polysaccharides and vitamins and have the potential to be used as ingredients in neutraceuticals. The antioxidant activity of crude 60% methanol extracts prepared from five Irish seaweeds, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus were examined using in-vitro assays and a cell model system to determine the antioxidant activity of the extracts and their ability to protect against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status in the human adenocarcinoma, Caco-2 cell line. To optimise the extraction of antioxidant compounds from seaweeds, an accelerated solvent extraction (ASE®) was used in combination with food grade solvents. The antioxidant activity of these extracts against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status was also assessed. Extracts that exhibited the highest antioxidant activity, A. nodosum (100% water and 80% ethanol extracts) and F. vesiculosus (60% ethanol extract) were selected as ingredients for incorporation into fluid milk and yogurt at concentrations of 0.25% and 0.5%. The addition of the seaweed extracts to milk and yogurt did not affect the pH or shelf-life properties of the products. Seaweed addition did however significantly influence the colour properties of the milk and yogurt. Yellowness values were significantly higher in yogurts containing F. vesiculosus at both concentrations and A. nodosum (80% ethanol) at the 0.5% concentration. In milk, the F. vesiculosus (60% ethanol) and A. nodosum (80% ethanol) at both the 0.25% and the 0.5% concentrations had higher greenness and yellowness values than the milk containing A. nodosum (100% water). Sensory analysis revealed that appearance and flavour governed the overall acceptability of yogurts with the control yogurt, and yogurts containing A. nodosum (100% water) were the most preferred samples by panellists. However, in the milk trial the perception of a fishy taste was the determining factor in the negative perception of milk. The unsupplemented control and the milk containing A. nodosum (100% water) at a concentration of 0.5% were the most overall accepted milk samples by the sensory panellists. The antioxidant activity of the extracts in milk and yogurt remained stable during storage as determined by the in-vitro assays. Seaweed supplemented milk and yogurt were also subjected to an in-vitro digestion procedure which mimics the human digestive system. The milk and yogurt samples and their digestates were added to Caco-2 cells to investigate their antioxidant potential however neither the undigested or digested samples protected against H2O2-induced DNA damage in Caco-2 cells.

Effects of synbiotics on cancer risk biomarkers

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world and second most common (behind lung cancer) in developed countries. In recent years there has been much interest in the potential use of prebiotics, probiotics and synbiotics in the prevention and treatment of CRC. We have previously shown that synbiotic consumption in Azoxymethane treated rats modulates the immune system, influences the genotoxic potential of caecal contents and reduces the number of colonic tumours compared to control rats who did not receive the synbiotic. The aim of the current study was to identify biomarkers suitable for use as cancer risk markers and as intervention markers. A second aim was to determine the influence of synbiotic consumption on cancer risk biomarkers such as in vivo colonic mucosal proliferation and genotoxic damage along with examining the genotoxic, cytotoxic and tumour promoting potential of faecal water (FW). Synbiotic consumption altered the composition of the gastrointestinal flora and reduced in vivo genotoxic damage and the genotoxic potential of FW in cancer and polyp subjects. Synbiotic consumption also reduced the proliferative activity in the colonic mucosa in polyp subjects. In both cancer and polyp subjects gene expression in the colonic mucosa was modulated in synbiotic consuming subjects. In this and other studies the activity of natural killer cells, the level of PGE2 in FW, IL-12 production by PBMCs, genotoxic damage in the colonic mucosa and the tumour promoting activities of FW have been identified as possible biomarkers of cancer risk. Future large scale studies investigating these parameters in healthy and diseased individuals are needed to confirm the suitability of these markers in assessing cancer risk and the role of synbiotics in modulating them.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Characterisation of the role of Fas in intestinal inflammation and cancer

Background: The role of Fas (CD95) and its ligand, Fas ligand (FasL/CD95L), is poorly understood in the intestine. Whilst Fas is best studies in terms of its function in apoptosis, recent studies suggest that Fas ligation may mediate additional, non-apoptotic functions such as inflammation. Toll like Receptors (TLRs) play an important role in mediating inflammation and homeostasis in the intestine. Recent studies have shown that a level of crosstalk exists between the Fas and TLR signalling pathways but this has not yet been investigated in the intestine. Aim: The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal cancer cells. Results: Treatment with TLR4 and TLR5 ligands, but not ligands for TLR2 and TLR9 increased the expression of Fas and FasL in intestinal cancer cells in vitro. Consistent with this, expression of Fas and FasL was reduced in the distal colon tissue from germ-free (GF), TLR4 and TLR5 knock-out (KO) mice but was unchanged in TLR2KO tissue, suggesting that intestinal cancer cells display a degree of specificity in their ability to upregulate Fas and FasL expression in response to TLR ligation. Expression of both Fas and FasL was significantly reduced in TRIF KO tissue, indicating that signalling via TRIF by TLR4 and TLR5 agonists may be responsible for the induction of Fas and FasL expression in intestinal cancer cells. In addition, modulating Fas signalling using agonistic anti-Fas augmented TLR4 and TLR5-mediated tumour necrosis factor alpha (TNFα) and interleukin 8 (IL)-8 production by intestinal cancer cells, suggesting crosstalk occurs between these receptors in these cells. Furthermore, suppression of Fas in intestinal cancer cells reduced the ability of the intestinal pathogens, Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8, suggesting that Fas signalling may play a role in intestinal host defence against pathogens. Inflammation is known to be important in colon tumourigenesis and Fas signalling on intestinal cancer cells has been shown to result in the production of inflammatory mediators. Fas-mediated signalling may therefore play a role in colon cancer development. Suppression of tumour-derived Fas by 85% led to a reduction in the tumour volume and changes in tumour infiltrating macrophages and neutrophils. TLR4 signalling has been shown to play a role in colon cancer via the recruitment and activation of alternatively activated immune cells. Given the crosstalk seen between Fas and TLR4 signalling in intestinal cancer cells in vitro, suppressing Fas signalling may enhance the efficacy of TLR4 antagonism in vivo. TLR4 antagonism resulted in smaller tumours with fewer infiltrating neutrophils. Whilst Fas downregulation did not significantly augment the ability of TLR4 antagonism to reduce the final tumour volume, Fas suppression may augment the anti-tumour effects of TLR4 antagonism as neutrophil infiltration was further reduced upon combinatorial treatment. Conclusion: Together, this study demonstrates evidence of a new role for Fas in the intestinal immune response and that manipulating Fas signalling has potential anti-tumour benefit.

The human papillomavirus and cancer: a multiphase study investigating preventative behaviours in young males

Background: The Human Papillomavirus (HPV) is one of the world’s most common sexually transmitted infections, and a causative factor of oropharyngeal, anal and penile cancers in males. Worldwide, an estimated 39,000 HPV-associated cancers occur each year in men. The highest rates of HPV infection are found in adults aged 18 to 28 years. Clinical evidence indicates that use of a condom in addition to obtaining the HPV vaccine provides the greatest protection from HPV infections. Aim: To explore young men’s attitudes, beliefs, and behavioural intention in relation to receiving the HPV vaccine and using a condom correctly and consistently. Collectively, both behaviours are linked to the prevention of HPV transmission and associated infections with HPV. Method: A multi- phase study, underpinned by the Theory of Planned Behaviour, involving a qualitative belief elicitation, pilot, and quantitative cross-sectional study was conducted. A belief elicitation (n=12) phase was used to generate items to include in a newly developed research instrument. Post pilot the research instrument was utilised in a cross sectional online survey to explore the attitudes, beliefs, and behavioural intention of young men (n= 359) with regard to receiving the HPV vaccine, and using a condom correctly and consistently. Data Collection: Data collection took place over a three month time frame. Male participants were recruited from a university in Southern Ireland via a student email system, as well as posting advertisements on numerous health, social and sports websites. Sample: Three hundred and fifty nine male participants aged 18- 28 years completed the online questionnaire. Data Analysis: Data were analysed using SPSS. Descriptive, correlational, multiple and hierarchical regression analysis were performed on the indirect and direct variables of the Theory of Planned Behaviour i.e. attitude, subjective norm, perceived behavioural control, and intention. Status variables were also included in descriptive analysis and hierarchical regressions. Findings are presented through text and graphical representation. Results: Alarming sexual health statistics identified that only 44.3% of participants always used a condom, and 78.6% never used a condom for oral sex. Furthermore, findings reveal that the constructs of the Theory of Planned Behaviour adequately measure male’s attitudes, beliefs and behavioural intention with regard to both behaviours. The Theory of Planned Behaviour has assisted in identifying how social pressures play an influential role in relation to males receiving the HPV vaccine. Attitudes presented as the most significant predictor of male’s intentions to use a condom correctly and consistently. Intention to perform both behaviours was identified as moderate to high. Conclusion: This study has contributed to the field of HPV research, as it is the first piece of research to explore preventative HPV behaviours i.e. receiving the HPV vaccine, and condom use, amongst young males, utilising the Theory of Planned Behaviour. A deeper understanding of young male’s attitudes, beliefs, and behavioural intention on this topic has been achieved. Additionally, a new robust research instrument has been constructed. Findings from this study will undoubtedly help in the implementation of the HPV vaccine in Ireland, as well as influence health promotion campaigns aimed at young males, addressing the topic of condom use.