AbstractSophorolipids (SL) are amphiphilic biosurfactant molecules consisting of a disaccharide sophorose with one fatty acid at the C1 position and optional acetylation at the C6’and C6” positions. They exist in a closed ring lactonic (LSL) or open acidic (ASL) structure. Sophorolipids are produced in crude mixtures in economically viable amounts by the yeast Starmerella bombicola and used in a variety of consumer products. Varying levels of anti- proliferative and anti-cancer activity of crude sophorolipid mixtures are described in a number of tumour cell lines in vitro. However, significant inter-study variation exists in the composition of SL species as well as other biologically active compounds in these mixtures, which makes interpretation of in vitro and in vivo studies difficult. It is hard to truly assess the anti-cancer action of SL due to the lack of in vivo studies. To date, only one other study evaluates the ability of a crude SL preparation to reduced tumour growth in a cervical cancer xenograft model. This thesis aims to evaluate the ability of two SL congeners, produced by Starmerella bombicola, which have been highly purified and well characterised against the colorectal cancer cell growth in vitro and the ability to regulate the development of precancerous polyps in vivo.
This thesis first evaluated and compared the biological activity of a 96% pure preparation of C18:1 LSL and a 94% pure C18:1 ASL preparation against CRC cells (HT29, HT115, HCT116, Caco2, LS180) and two normal cell lines CCD-841-CoN (colonic epithelial) and MRC5 (lung fibroblast) in vitro by assessing their ability to modulate cell viability via a MTT assay and the regulate the metastatic properties of these cells by assessing changes to migration and invasion in culture. The overall consensus of the data suggest that LSL induce cytotoxicity in CRC cells but also induces cytotoxity in the normal colonic CCD-841-CoN iv cells at lower concentrations; results are detailed further in chapter three. In comparison, ASL reduced the viability of the CRC cell lines at low concentrations but proved non-toxic to the normal colonic cell line; results of these studies are detailed further in chapter four.
In the latter portions of chapter three and four respectively, the biological activity of both congers were assessed in wt c57 mice and Apcmin+/- mice, a recognised animal model of Familial adenomatous polyposis (FAP) by first ensuring both LSL and ASL were well tolerated at doses of 0.5, 5 and 50mg/kg when administered orally. Next the ability of SL congeners to mediate adenomatous polyp growth was measured by counting the number of polyps present in the intestinal tract of Apcmin+/- after 70 days of SL treatment compared to the vehicle-only treated control mice. In addition, parameters such as spleen weight and haematocrit levels were also measured as a method to evaluate the ability of SL to regulate the secondary side-effects associated with reducing the life-span of the Apcmin+/- . In summary, LSL resulted in exacerbated polyp growth in Apcmin+/- with a further decrease in haematocrit in addition to splenomegaly. ASL had no effect on tumour number of size but increased haematocrit levels and reduced the spleen size of the treated Apcmin+/- mice compared to vehicle control.
The final experimental chapter of this thesis explored the possible mechanisms of action of SL in vitro. The CRC cancer cell line HT29 was used to assess SL induced alterations to lipid raft expression, changes to tight junctions and SL congener specific changes to mitochondrial membrane potential and reactive oxygen species production in vitro, as described in chapter five.
In summary, contrary to current literature, purified ASL appears to have an advantage over the LSL as a potential therapeutic agent due to its ability to selectively reduce viability of v CRC cells without inducing toxicity at equivalent doses in normal colonic epithelial and lung fibroblasts form in vitro. Although ASL did not disrupt the tumour growth in the Apcmin+/- mice, they improved the haematocrit levels and decreased spleen size which could result in an increase in life-span to these mice as they are known to succumb to the effects of intestinal bleeding in the 5th – 6 th month of life. To date, this is the first study to investigate the effects of a highly purified LSL and ASL preparation across a wide range of CRC cells in vitro and the first study to investigate the anti-cancer of purified preparation in vivo.
|Date of Award||Oct 2017|
|Sponsors||Department of Education and Learning (DEL)|
|Supervisor||Christopher Mitchell (Supervisor) & Ibrahim Banat (Supervisor)|
- Colon FAP
- Animal Model