Bridge, Jack (2019) ABC Transporters and the creation of a tumour-conducive microenvironment in neuroblastoma. Masters thesis, University of Huddersfield.

ABC transporters have long been implicated in the promotion of cancer survival due to their role in cytotoxic drug efflux and conferring MDR. The broad substrate specificity associated with ABC transporters, particularly in the ABCC subfamily, is however implicating ABC transporters in the promotion of carcinogenesis independent of drug efflux. Eicosanoids including PGE2 and cysteinyl leukotrienes are among the substrates transported by ABCC transporters and are main players in the body’s natural inflammatory response and more importantly, tumour-promoting inflammation.

Neuroblastoma cell lines overexpressing the MycN oncogene are documented to heavily expand their ABC expression profile, with upregulations seen in both ABCC1 and ABCC4. This project aims to assess how the expression of certain ABCs like ABCC1 and 4 affects extracellular concentrations of eicosanoids by inhibiting their transport, at the same time assessing how ABC expression affects the migratory potential of cells and potentially implicating PGE2 and cysteinyl leukotrienes in the more aggressive phenotypes demonstrated by NB cell lines.

cDNA converted from the RNA of 5 different NB cell lines (3 MycN amplified, 2 non-MycN amplified) was used to run qPCR to determine their ABC transporter expression profile and explore the effects of MycN amplification on the expression of ABC transporters involved in eicosanoid efflux. Wound- healing assays were used to determine the migratory potential of different NB cell lines under pharmacological inhibition of transporters ABCB1, C1 and C4.

The effects of ABC inhibition on eicosanoid efflux using competitive ELISA was also examined to explore potential associations with ABC transporter expression and eicosanoid efflux.

KELLY and SK-N-BE(2)c cells showed the ABC expression patterns previously documented to be associated with MycN amplification, showing increases in ABCC1 and 4 and a downregulation of ABCC3. IMR32 cells did not follow this trend but had the most aggressive migratory phenotype under basal conditions. ABC inhibition had no effect on the migration of NB cells, regardless of MycN amplification.

The expression patterns of the ABC transporters considered in this project did not appear to have a major effect on the efflux of eicosanoids or cAMP, contradicting the original hypothesis that ABC-mediated eicosanoid efflux is a major player in enhancing the migration and invasiveness of NB cell lines, accounting for the more aggressive phenotype in MycN amplified cell lines. This does not conclude however that ABC transporter expression is not associated with NB cell behaviour as other carcinogenic hallmarks remain to be explored in this field. The redundancy in substrate specificity of some ABC transporters, namely ABCC1 and ABCC4 with their shared specificity to PGE2, needs to be addressed in future studies with the use of multiple inhibitors to more accurately restrict efflux of eicosanoids and better demonstrate their impact on migration. When focusing on key inflammatory eicosanoids such as PGE2 and cysteinyl leukotrienes, ABCCC1 and C4 are the most important to target as they are the main mediators of eicosanoid efflux and are currently not well understood.

FINAL THESIS - BRIDGE.pdf - Accepted Version
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