Screening human cancer pathways for a role of Aflatoxin b1

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September, 2015
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Aflatoxin B1 (AFB1) has been shown to negatively affect the functions of major body organs such as the liver, kidney, spleen and heart, as well as decreasing cellular immunity and causing cancers, primarily hepatocellular carcinoma. Some studies have suggested that mutation in the codon 249 of p53 tumour suppressor gene is a key event in AFB1 induced carcinogenesis but the effects of AFB1 on other cancer pathways are not known. The aim of the study was to screen 45 cancer pathways for a role of AFB1 using human embryonic kidney cells (HEK 293) as a model system. Cytotoxicity was determined by treating HEK 293 cells with increasing concentrations of AFB1 for 24, 48 and 72 hours and the viability of cells was evaluated by an MTS based assay. It was established that increasing concentration of AFB1 killed cells in a dose dependent manner with 32μM being established as the working concentration. The cells were then reversed transfected with pre-coated pathway reporters in Cignal Finder 45-pathway reporter array for 24 hours and treated with or without 32μM of AFB1 for 24 hours and harvested. The influence of AFB1 on the pathways was then determined by measuring the luciferase activity of the pathway reporters using dual luciferase reporter gene assay. Validation of data from the Cignal Finder 45-pathway reporter array was achieved by measuring the influence of AFB1 on interferon (IFN) induced signal transduction pathway in an independent assay. The results of Cignal Finder 45-pathway reporter array showed that AFB1 differentially modulates multiple pathways. Key pathway reporters up-regulated included AARE, ARE, ATF-6, GRE, MTF-1, ISRE, NFκB and PAX-6 whereas p53, FoxO, NFAT, PPAR, SP-1 and STAT3 were down-regulated. Results from the validation experiment showed that AFB1 up-regulated IFN induced signal transduction which was consistent with that of Cignal Finder 45-pathway reporter arrays. These results show that AFB1 could cause cancer by deregulating multiple pathways aside the p53 pathway suggesting that further studies should be done to establish the exact point of influence of AFB1 on these pathways.
A thesis submitted to the Department of Clinical Microbiology, Kwame Nkrumah University of Science and Technology, in partial fulfillment of the requirements for the degree of Master of Science (Clinical Microbiology),2015.