The effects of sulforaphane on normal and cancerous epithelial cells

Alqurashi, Yaser (2017) The effects of sulforaphane on normal and cancerous epithelial cells. Doctoral thesis, University of East Anglia.

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The cytoskeleton plays vital roles in many cell functions and any disorder could lead to different diseases such as cancer, which causes uncontrolled cell division, cell motility and invasion. Cell migration is important for developmental morphogenesis, tissue repair and tumour metastasis. During the migration process, microtubule organisation and dynamics play important roles, and are regulated by Plus-end Tracking (+TIP) proteins. A better understanding of cell migration mechanisms could lead to an efficient treatment for invasive cancer cells such as pancreatic cancer. Sulforaphane (SFN) is a potential treatment for different cancer types with various possible means of action. Studies have indicated that SFN has the potential to slow down the progression of cancer, promote apoptosis and suppress cell proliferation. However, how SFN affects microtubule dynamics, stability and organisation is poorly understood.
This project investigated the effect of SFN on microtubule organisation and dynamics and the impact on cell migration. Certain pancreatic cancers show up-regulation of the end-binding protein EB2 and the tubulin deacetylase HDAC6 which both influence microtubule dynamics and stability and thus also cell migration. Other cancers such as breast cancer have reported an up-regulation in EB1. This project therefore focused on the effects of SFN on EB1, EB2 and HDAC6 expression and localisation and the consequences for cell migration.
The impact of SFN on normal (ARPE-19) and cancerous (Panc-1) epithelial cell migration was assessed by live time-lapse imaging of sparsely seeded cells. Microtubule and actin filament organisation was assessed to observe the effects of SFN on the treated cells. The impact of SFN on EB and HDAC6 localisation and expression was determined by immuno-labelling. Experiments involving SFN treatment with or without functional inhibition of HDAC6 (with the HDAC6 inhibitor tubacin) were carried out and their effect on cell migration was investigated. The effect of SFN on microtubule dynamics and stability was assessed by analysing live time-lapse GFP-CLIP-170 dynamics. Microtubule post-translational modifications were studied by immuno-labelling in SFN treated cells. Focal adhesion area and dynamics were also assessed by FRAP and immuno-labelling in SFN treated cells.
SFN treatment caused a dramatic decrease in the speed of random migration and cell area in ARPE-19 cells. A marked co-alignment between microtubules and actin filaments, increased EB1 decoration of the microtubule lattice and an apparent increase in cytoplasmic EB2 were also observed upon SFN treatment. However, SFN treated Panc-1 cells revealed no significant decrease in the speed of random cell migration. Interestingly, a combination of tubacin and SFN caused a significant decrease in the speed of cell migration. Analysis of microtubule dynamics in GFP-CLIP-170 expressing cells revealed that SFN treated cells possessed less dynamic microtubules. There was also evidence of SFN inducing microtubule stability.
The results suggest that SFN or a combination of tubacin and SFN could be promising treatments for cancer. Moreover, these results can provide a better understanding of the effects of SFN on the organisation of the cell cytoskeleton. Treatment with SFN resulted in interesting changes in EB and HDAC6 localisation, which may provide potential targets for effective cancer treatments.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 4971 not found.
Date Deposited: 12 Oct 2017 14:04
Last Modified: 31 Oct 2020 01:38

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