Validation of shellfish isolates (glycosaminoglycans, GAGs) for development as a novel anti-tumour therapy for children : GAG action on lymphocytes/ T-regulatory cells

Jones, CF 2019, Validation of shellfish isolates (glycosaminoglycans, GAGs) for development as a novel anti-tumour therapy for children : GAG action on lymphocytes/ T-regulatory cells , PhD thesis, University of Salford.

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Abstract

Introduction: Leukaemia is cancer of white blood cells (WBCs) mainly affecting children. Defective WBCs are prominent in blood and cannot provide immune protection. Although chemotherapy treatments are effective, they have many adverse effects which are magnified in children, with treatment proving fatal for around 10-20% of children undergoing treatment. T-regulatory cells regulate inflammatory responses; in cancer raised levels of T-regulatory cells have been linked with immune evasion of cancer cells. Understanding chemotherapy effects on Treg populations is key to predicting therapy effectiveness. Ongoing research at KidsCan has identified the anti-tumour properties of novel glycosaminoglycan (GAG) compounds isolated from shellfish. However the effect of GAGs on normal lymphocyte populations is still unknown, identifying this action is essential in order to evaluate any potential use of GAGs as a cancer chemotherapy treatment in children.
Aim: In order to further understand the potential use of GAGs for clinical applications the aim of this research is to evaluate T-cell and B-cell responses (in healthy lymphocytes) to GAG treatment and to compare them to control cancer cell lines (MOLT-4, K562 and U698).
Methods: Phase 1 of the research isolated GAGs from cockles and whelks. Peripheral blood mononuclear cells (PBMCs) were extracted in bulk from whole blood from the NHS donor service. GAG isolates were tested for activity on cell growth using MTT assay on PBMCs (naïve and activated) and three cancer cell lines (MOLT-4, K562, U698). Phase 2 tested the GAG isolates for their potency via a flow cytometric annexin V/ PI apoptosis assay on the cell lines and PBMCs both activated using either PHA or PMA/ionomycin and naïve. Phase 3 studies evaluated individual FPLC separated GAG fractions to establish potentially potent domains of the compounds. This work also focussed on T-regulatory cell (Treg) responses to the GAGs to provide an insight to their potential modulation of a population of cells associated with cancer progression.
Results: The MTT assays identified that both GAG extracts had a profound effect on cancer cell growth, with cell viability inhibitions of up to 90% and IC50 values ranging between 0.7 µg/ml and 12 µg/ml. This study also indicated that GAG extracts have little effect on the viability of healthy lymphocytes (PBMCs) identifying the potential of GAGs as a therapeutic treatment for cancer. MTT assays aided with the design of the apoptosis assays. Apoptosis assays indicated that cancer cells responded to GAG extract treatment via the induction of apoptosis and identified which cell types were most at risk of being targeted in healthy lymphocyte populations (stimulated and unstimulated), they also identified the whelk extract as being the more effective GAG treatment. The Treg assays indicate that decreases in the cell population may be an additional benefit of the GAG compounds through reducing the risk of tumour progression. However further work with Treg populations in healthy lymphocytes would help to prove this principle and future studies would be useful to further optimise the fraction assays.

Item Type: Thesis (PhD)
Contributors: Smyth, LJC (Supervisor) and Pye, DA (Collaborator)
Schools: Schools > School of Environment and Life Sciences > Biomedical Research Centre
Funders: KidsCan
Depositing User: Chloe Francesca Jones
Date Deposited: 09 Oct 2019 09:00
Last Modified: 09 Nov 2019 02:30
URI: http://usir.salford.ac.uk/id/eprint/52261

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